Phage-mimicking nanoparticles and polyethylenimine metal implant coatings

Abstract

The disclosure provides for coatings disposed on a surface of a medical implant, where the coating includes a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: a silica core, silver coated gold nanospheres disposed on a surface of the silica core, and plurality of carboxy thiol functionalized (CTF)-polyethylenimine (PEI) polymers conjugated to the silver nanospheres, where the CTF-PEI polymers include a CTF moiety and branched and / or linear PEI moieties.

Description

[0001] PHAGE-MIMICKING NANOPARTICLES AND

[0002] POLYETHYLENIMINE METAL IMPLANT COATINGS

[0003] RELATED APPLICATIONS

[0004] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 684,254 filed August 16, 2024, the entire disclosure of which is hereby incorporated by reference in its entirety.

[0005] BACKGROUND OF THE INVENTION

[0006] The demand for metal implant surgery is rapidly increasing due to the fast-growing and ageing population, posing an increased burden on the health care system. The annual report of the American Joint Replacement Registry in 2021 stated that there has been an increase of 18.3% in total procedural volume compared to 2020 in the US, with over 2.4 million hip and knee surgeries. Metal implants are used in various medical procedures, such as joint replacement, fracture fixation or dental settings to improve an impaired function or fully replace missing functions. Made of non-biological material, it is vital for the implants to be biocompatible, and for the long-term success of the implant, the material needs to be able to sustain the conditions in the physiological environment while the biological system itself also needs to accept the implant material.

[0007] One of the greatest concerns regarding metal implant surgery failure is the implant- associated infections caused by various pathogenic bacteria, such as 5. aureus, K. pneumoniae and P. aeruginosa, with the most common bacteria causing the infections being Staphylococci. Depending on the type of implant surgery, the probability of developing an infection is 2-30%. Antibiotics and their slow release from the metal implant surface post-surgery remain the most common treatment for preventing infection. Concerns remain about the development of antibiotic resistance or infections of already resistant bacteria strains such as .S'. aureus and P. aeruginosa with antibiotics being the most common treatment before surgery.

[0008] Other concerns in implant related infections are the formation of biofilms. Bacteria such as .S'. aureus and P. aeruginosa are able to adhere to metal implant surfaces, forming a biofilm which can lead to increased antibiotic-resistant, and other complications that lead to implant failure. Biofilm formation allows the bacteria to adhere to the implant surface and form communities that are enclosed by bacteria-produced exopolysaccharide matrix, making the bacteria within the biofilm metabolically inactive. The matrix created from biofilm formation can prevent antibiotics from penetrating into the bacteria cells, leading to severe bacterial infections with antibiotic resistance up to 1000 times higher than in systemic bacteria. With concerns about

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[0011] 501.112WO1 biofilm formation, research is being done to use localized antibiotics and optimize their efficacy. However, these treatments do not resolve concerns about the development of antibiotic resistance.

[0012] Accordingly, there is a need for a medical device coating free of antibiotics that reduces or eliminates bacterial populations on the surface of the medical implant to which the coating is adhered. This invention satisfies these needs.

[0013] SUMMARY OF THE INVENTION

[0014] Incidence of implant-associated infections of bacteria with antibiotic resistance will rise as the number of joint implant surgeries increases due to an aging population. This rise in implant associated antibiotic resistant infections shows the need to develop methods that prevent infections without antibiotics. Here, Applicants utilize previously designed phage-mimicking nanoparticle (PhaNP) to create an antimicrobial metal implant coating by combining PhaNPs with antimicrobial polymers. Metal implant coupons were coated with PhaNPs and different polyethylenimine polymers: branched 11-MUA-PEI 600, branched 11-MUA-PEI 1800, and linear 11-MUA-PEI 2500. The different surface modifications were tested for antibacterial properties in vitro against 5. aureus USA300 and P. aeruginosa FRD1, and E. coli K12.

[0015] The polyethylenimine conjugated PhANPs showed significantly less viable and adhering bacteria against .S'. aureus USA300 compared to unmodified and PhANPs modified surfaces. The PhANPs and the polyethylenimine conjugated PhANPs showed significantly less viable and adhering P. aeruginosa FRD1 compared to the unmodified surface, and similarly, for E. coli K12. In vitro biocompatibility tests were completed using HaCaT human skin keratinocyte and MG-63 osteoblast cell lines, and every modification showed very low cytotoxicity of against either of the mammalian cell lines, proving our design is highly biocompatible. Therefore, Applicants have developed a novel antibiotic-free antibacterial coating for metal implants, displaying high antibacterial effects towards two clinically relevant bacteria strains, while having low cytotoxicity towards mammalian cells.

[0016] Accordingly, the disclosure provides for a coating disposed on a surface of a medical implant, the coating comprising a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: a silica core; one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of carboxy thiol functionalized (CTF)-polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the plurality of CTF-PEI polymers comprises a plurality of CTF moieties and a plurality of branched and / or linear PEI moieties. In some embodiments, the one or more CTF moieties comprises - CO(CI-C2O)SH or a -CO(thiophenyl). In some embodiments, CTF moieties comprise one or more

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[0019] 501.112WO1 of 11-mercaptoundecanoic acid, 3-mercaptopropionic acid, mercaptoacetic acid, 4- mercaptobenzoic acid, 3-mercaptobenzoic acid, 6-mercaptohexanoic acid, and thio-salicyclic acid. In some certain embodiments, the plurality of CTF moieties comprise 11- mercaptoundecanoic acid to form a plurality of 11-MUA-PEI polymers.

[0020] In some embodiments, the plurality of the CTF-PEI polymers comprise a molar ratio of CTF moieties to PEI of about 1:5 to about 5:1. In some embodiments, the plurality of the CTF- PEI polymers comprise a molar ratio of CTF moieties to PEI of about 1:1 to about 1:5. In some embodiments, the plurality of the CTF-PEI polymers comprise a molar ratio of CTF moieties to PEI of about 1:1 to about 1:3.

[0021] In some embodiments, each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 250 Da to about 3500 Da. In some embodiments, each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 500 Da to about 3000 Da. In some embodiments, each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 500 Da to about 2000 Da. In some embodiments, each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da or about 1800 Da. In some embodiments, each of the plurality of branched and / or linear PEI moieties is a linear PEI polymer comprising an average molecular weight of about 2000 to about 3000 Da. In some embodiments, each of the plurality of branched and / or linear PEI moieties is a linear PEI polymer comprising an average molecular weight of about 2500 Da.

[0022] In some embodiments, the plurality of 11-MUA-PEI polymers comprises at least two distinct CTF-PEI polymer populations, wherein a first CTF-PEI polymer population comprises CTF moieties and a branched PEI moiety having an average molecular weight of about 600 or about 1800, and a second CTF-PEI polymer population comprises CTF moieties and linear PEI moiety having an average molecular weight of about 2500 Da.

[0023] The disclosure also provides for methods of coating a surface of a metal medical implant comprising the steps of: i) polishing the surface of the metal medical implant; ii) silanizing the surface of the metal medical implant; iii) adhering a plurality of phage mimicking nanoparticles (PhaNPs) to the silanized surface; iv) modifying a surface of the PhaNPs adhered to the surface of the medical implant with a plurality of carboxy thiol functionalized (CTF)-PEI polymers, wherein the plurality of CTF-PEI polymers comprises one or more CTF moieties and one or more branched and / or linear PEI moieties; and wherein the PhaNPs comprise: a) a silica core; and b) one or more silver coated gold nanospheres disposed on a surface of the silica core; and wherein the surface of the PhaNP comprises the one or more silver coated gold nanospheres.

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[0026] 501.112WO1 In some embodiments, a coating that is disposed on a surface of a medical implant, the coating comprising a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i. a silica core; ii. a plurality of silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (ll-MUA)-polyethylenimine (PEI) polymers conjugated to the plurality of silver coated gold nanospheres, wherein the 11- MUA-PEI polymers comprise one or more 11-MUA moieties and one or more branched and / or linear PEI moieties.

[0027] These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0030] Fig. 1A-B. (A) A visual representation of the PhaNP synthesis process, (B) metal coupon modification process.

[0031] Fig. 2A-C. Nanoparticle characterization. (A) i) TEM image of SiNPs before surface modifications with Au and Ag and ii) the size distribution of SiNPs with an average diameter of 22.91 nm ± 2.74 nm. (B) i) TEM image of PhaNPs used for metal coupon surface modification and ii) the size distribution of PhaNPs with an average diameter of 24.38 nm ± 2.43 nm. (C) The elemental composition of the PhaNPs was confirmed by energy-dispersive X-ray spectrum (EDXS) by detecting distinct energy peaks for Si (Ka = 1740 eV), O (Ka = 525 eV), Au (Ma = 2123 eV, La = 9.713 keV) and Ag (Ka = 22.163 keV, La = 2.983 keV). Mg, Na and Cl peaks are present due to the solvent used (PBS). The bar size is 1000 nm.

[0032] Fig. 3A-D. Metal implant coupon characterization. (A) SEM of unmodified metal implant confirming polished surface. The size bar is 50 pm. (B) The elemental composition of the metal implant coupon was confirmed by energy-dispersive X-ray spectrum (EDXS) by detecting distinct energy peaks for Fe (Ka = 6.398 keV, La = 0.705 keV), Ni (Ka = 7.471 keV, La = 0.851 keV),

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[0035] 501.112WO1 Cr (Ka = 5.411 keV, La = 0.573 keV) and Mo (Ma = 17.441 keV, La = 2.293 keV). (C) SEM of PhaNP and polymer-modified metal coupon confirming the presence of PhaNPs on the metal surface. The size bar in the inset is 50 pm and the size bar on the main panel is 500 pm. (D) FTIR of the modified metal coupon surface (MUA-LP) compared to the unmodified metal confirmed the presence of SiCL of the PhaNPs at 1200 cm'1and the polymer at 1650 cm'1. Open circle = metal and open square = metal-PhaNP@Cl 1-LP.

[0036] Fig. 4A-F. Antibacterial Activity of Varying Metal Implant Surface Modifications (A)- (B) 5. aureus USA300 LIVE / DEAD assay and SEM. (C) Color segmentation results of 5. aureus USA300 LIVE / DEAD assay images (n=3) with one-way ANOVA testing between each surface modification. Standard deviations of the average % area are used as error bars. * denotes significance in comparison to unmodified surface, ** denotes significance in comparison to PhANPs surface. (D)-(E) P. aeruginosa FRD1 LIVE / DEAD assay and SEM. Open circle = live, open triangle = dead, open square = empty area / unmodified surface. (F) Color segmentation results of P. aeruginosa FRD1 LIVE / DEAD assay images (n=3) with one-way ANOVA testing between each surface modification. Standard deviations of the average % area are used as error bars. * denotes significance in comparison to unmodified surface; Open circle = live, open triangle = dead, open square = empty area / unmodified surface).

[0037] Fig. 5A-D. Biocompatibility of Metal Implants with Various Surface Modifications. (A)- (B) HaCaT cells LIVE / DEAD assay and SEM. (C) Color segmentation results of HaCaT LIVE / DEAD assay images (n=3) with one-way ANOVA testing between each surface modification. Standard deviations of the average % area are used as error bars. Open circle = live, open triangle = dead, open square = empty area / unmodified surface. (D)-(E) MG63 cells LIVE / DEAD assay and SEM. (F) Color segmentation results of MG63 LIVE / DEAD assay images (n=3) with one-way ANOVA testing between each surface modification. Standard deviations of the average % area are used as error bars. * denotes significance in comparison to PhANPs + MUA-PEI 600 + MUA-LP surface; Open circle = live, open triangle = dead, open square = empty area / unmodified surface).

[0038] Fig. 6. In unmodified coupons, there were live and dead cells and SEM confirmed the presence of multiple bacteria in high magnification. PhaNP modified coupons killed bacteria but bacteria also stayed on coupon after death. PhaNP @C 11-600 polymer modified coupons killed bacteria and bacteria debris seemed to stay on coupon after death. PhaNP@Cl 1-1800 polymer modified coupons killed bacteria and there was minimal bacteria debris on coupon.

[0039] Fig. 7. PhaNP@Cl l-linear polymer modified coupons killed bacteria and no discernable fluorescence signal was noted on LIVE dead assay. No discernable bacterial debris on the 5

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[0041] 501.112WO1 coupons. Similarly, PhaNP@Cl 1-linear polymer+1800 polymer modified coupons killed bacteria and no discernable fluorescence signal was noted on LIVE dead assay. No discernable bacterial debris on the coupons.

[0042] Fig. 8. Approximate Correlation Matrix for % Live Area. To contextualize these findings, an approximate correlation matrix for % Live Area (bacterial vs. mammalian cells) illustrates the inverse relationship between bacterial viability and eukaryotic cell attachment across various coatings. Negative correlations (e.g., -0.70 between P. aeruginosa and MG63) indicate that coatings supporting high bacterial viability often show low osteoblast-like cell attachment, and vice versa. Positive correlations (0.60 between MG63 and HaCaT) suggest that a coating favoring one mammalian cell line often favors another, consistent with a shared need for conducive surface chemistry and moderate hydrophilicity. Overall, these correlations reinforce the conclusion that coating strategies reducing bacterial adhesion simultaneously enhance mammalian cell compatibility — an ideal outcome for biomedical implants.

[0043] DETAILED DESCRIPTION

[0044] Definitions.

[0045] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001 or Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N.Y. (1991). General laboratory techniques (DNA extraction, RNA extraction, cloning, PCR amplification, cell culturing, etc.) are known in the art and described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, 2012.

[0046] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Lurther, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure,

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[0049] 501.112WO1 moiety, or characteristic with other embodiments, whether or not explicitly described.

[0050] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0051] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring.

[0052] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0053] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0054] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all 7

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[0056] 501.112WO1 possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units is also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or average molecular weight) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into subranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0057] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number 2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, ..., 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above.

[0058] The recitation of a), b), c), ...or i), ii), iii), or the like in a list of components or steps do not confer any particular order unless explicitly stated.

[0059] Dosing conversions between animals and humans are known in the art, and described, for example, by Nair et al., J Basic Clin Pharma 2016;7:27-31.

[0060] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of 8

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[0062] 501.112WO1 the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0063] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0064] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open- ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein.

[0065] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0066] An "effective amount" refers to an amount effective to bring about a recited effect, such as an amount necessary to form products in a reaction mixture. Determination of an effective amount is typically within the capacity of persons skilled in the art, especially in light of the detailed disclosure provided herein. The term "effective amount" is intended to include an amount of a compound or reagent described herein, or an amount of a combination of compounds or reagents described herein, e.g., that is effective to form products in a reaction mixture. Thus, an "effective amount" generally means an amount that provides the desired effect.

[0067] The terms "treating", "treat" and "treatment" include (i) inhibiting the disease, pathologic or medical condition or arresting its development; (ii) relieving the disease, pathologic or medical 9

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[0069] 501.112WO1 condition; and / or (iii) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can include lowering, stopping, or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical and therapeutic administration, as appropriate.

[0070] The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.

[0071] As used herein, terms "subject" or "patient" is used interchangeably to refer to an animal (e.g., birds, reptiles, and mammals). In a specific embodiment, a subject is a bird. In another embodiment, a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In certain embodiments, a subject is a non-human animal. In another embodiment, a subject is a human.

[0072] The term “medical device” as used herein is intended to extend broadly to all devices used in the medical field, including stents, catheters, various implants and the like regardless of the material from which it is fabricated. References herein to medical devices and other medical references are understood to also include veterinary devices and applications.

[0073] As the number of joint implant surgeries increases due to the aging population, infections from antibiotic -resistant bacteria are expected to rise. This highlights the need for infectionpreventing methods that don't rely on antibiotics. In response, researchers have developed a new antibacterial coating for metal implants using specially designed nanoparticles (PhaNP) combined with antimicrobial polymers. We tested various coatings on metal surfaces against two harmful bacteria strains, .S'. aureus USA300 and P. aeruginosa FRD1, and found that the new coatings significantly reduced the number of bacteria compared to uncoated surfaces by exhibiting contact killing of bacteria. Additionally, the coatings were tested on human skin and bone cells and showed very low toxicity, proving them to be safe for human use. Thus, this new coating is an effective and safe alternative to antibiotics for preventing infections on metal implants.

[0074] Accordingly, disclosed herein are surface coatings, metal medical implants comprising a surface coating, and methods of adding a surface coating to a metal medical implant. Generally, the surface coating comprises a plurality of phage mimicking nanospheres (PhaNPs) functionalize with polymers comprising carboxy thiol functionalized polyethyleneimines, where the polyethyleneimines comprise branched or linear polyethyleneimine moieties. Preferably, the

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[0077] 501.112WO1 carboxy thiol functionalized-PEI polymers are conjugated to the surface of the phage mimicking nanoparticles.

[0078] As used herein, the term “polyethylenimine” or “PEI” refers to a polymer composed of repeating ethyleneimine units (-CH2CH2NH-), available in both branched and linear forms (see U.S. Patent No.12,059,654 to Spulber et al. Jager et al., Chem Soc Rev. 2012 Jul 7;41(13):4755- 67). The term “11-Mercaptoundecanoic acid” or “11-MUA” refers to a compound having the formula C11H22O2S.

[0079] In some embodiments, a medical implant coating disposed on a surface of the medical implant comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i. a silica core; ii. one or more gold nanospheres disposed on a surface of the silica core; iii. one or more silver nanospheres surrounding the one or more gold nanospheres, thereby forming a surface of the PhaNPs; and a plurality of carboxy thiol functionalized (CTF)- polyethylenimine (PEI) polymers conjugated to the surface of the PhaNPs, wherein the plurality of the CTF-PEI polymers comprises a plurality CTF moieties and one or more branched and / or linear PEI moieties.

[0080] In some embodiments, the carboxy thiol functionalized polyethyleneimines are represented by Formula I: wherein,

[0081] R1, R2, and R3are each independently each y is each independently 0-20.

[0082] In some embodiments, y is about 1 to about 18, about 5 to about 15, about 10 to about 15, or about 8 to about 12. In some embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, 15, 16, 17, 18, 19, or 20.

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[0085] 501.112WO1 In some embodiments, the CTF moiety comprises one or more of 11-mercaptoundecanoic acid, mercaptoacetic acid, 3-Mercaptopropionic acid, 2-mercaptobenzoic acid, 4- Mercaptobenzoic acid, 3 -Mercaptobenzoic acid, 4-Mercaptobutyric acid, 5 -Mercaptopentanoic acid, Mercaptohexanoic acid, thiosalicylic acid, Thiophenecarboxylic acids (e.g., 2-mercapto-3- phenylpropionic acid), Cysteine, Homocysteine, and Penicillamine. The amines on the PEI can also be conjugated to thiol bearing organic molecules through reductive amination with an mercaptoaldehydes (e.g.,3-Mercapto-2-methylpentanal, 3-Mercaptohexanal,

[0086] Mercaptoacetaldehyde) or mercaptoketone (e.g., 4-Mercapto-4-methyl-2-pentanone). Any NHS esters of CLICK reagents (e.g., DBCO-NHS, alkyne NHS ester, NHS Azide) can be utilized to introduce CLICKable functional groups on PEI by linking the CLICK reagent to the amine group of PEI through an NHS ester mediated amide bond formation. The complementary CLICK group on a thiol linker (e.g., Azide thiol to CLICK with DBCO, Azide thiol to CLICK alkyne through copper catalyzed reaction, DBCO-PEG-SH or DBCO-propanamide-thiol or DBCO- Thiol can be CLICKed to Azide) can be utilized to introduce a thiol group on the PEI as well. Isothiocyanates also react with amines. Thiol isothiocyanates such as EITC-PEG thiols (MW 200 or 400 or 600 or 800 or 1000 such as described at www.biochempeg.com / product / FITC-PEG-SH.html) can also be utilized to introduce thiol functional group on PEI.

[0087] In some embodiments, the CTE moiety comprises one or more of 11-mercaptoundecanoic acid, 3-Mercaptopropionic acid, mercaptoacetic acid, 4-Mercaptobenzoic acid, 3- Mercaptobenzoic acid, 6-Mercaptohexanoic acid, and thiosalicylic acid.

[0088] In some embodiments, the one or more CTF moieties comprises a -CO(CI-C2O)SH or a - CO(thiophenyl). In some embodiments, the -CO(CI-C2O)SH, when present, is 11- mercaptoundecanoic acid, mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, or 6-mercaptohexanoic acid; and the -CO(thiophenyl), when present, is thiosalicylic acid.

[0089] In some embodiments, a medical implant coating disposed on a surface of the medical implant comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i. a silica core; ii. one or more gold nanospheres disposed on a surface of the silica core; iii. one or more silver nanospheres surrounding the one or more gold nanospheres, thereby forming a surface of the PhaNPs; and a plurality of carboxy thiol functionalized (CTF)- polyethylenimine (PEI) polymers conjugated to the surface of the PhaNPs, wherein the plurality of the CTF-PEI polymers comprises a plurality CTF moieties and one or more branched and / or linear PEI moieties, wherein the plurality of CTF moieties is selected from the group consisting

[0090] 12

[0091] ND 25-001

[0092] 501.112WO1 of 11-mercaptoundecanoic acid, mercaptoacetic acid, 3-Mercaptopropionic acid, 4- Mercaptobenzoic acid, 3-Mercaptobenzoic acid, 6-Mercaptohexanoic acid, and thiosalicylic acid.

[0093] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core, forming a surface of the PhaNPs; and a plurality of 11- mercaptoundecanoic acid (1 l-MUA)-polyethylenimine (PEI) polymers conjugated to a surface of the PhaNPs, wherein the plurality of the 11-MUA-PEI polymers comprises one or more 11-MUA moieties and one or more branched and / or linear PEI moieties.

[0094] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the plurality of the 11-MUA-PEI polymers comprises one or more 11-MUA moieties and one or more branched PEI moieties.

[0095] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the plurality of the 11-MUA-PEI polymers comprises one or more 11-MUA moieties and one or more linear PEI moieties.

[0096] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the plurality of the 11-MUA-PEI polymers comprises one or more 11-MUA moieties and one or more branched and / or linear PEI moieties, and wherein the plurality of the 11-MUA-PEI polymers comprise a molar ratio of 11-MUA to PEI of about 1 : 1 to about 1:5.

[0097] In some embodiments, a medical implant coating disposed on a surface of the medical implant a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i. a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and 13

[0098] ND 25-001

[0099] 501.112WO1 a plurality of 11-mercaptoundecanoic acid (l l-MUA)-polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the PEI comprises one or more branched PEI moieties, wherein each of the one or more branched PEI moieties comprise an average molecular weight of about 600 Da.

[0100] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the PEI comprises a one or more branched PEI moieties, wherein each of the one or more branched PEI moieties comprise an average molecular weight of about 1800 Da.

[0101] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the PEI comprises one or more linear PEI moieties, wherein each of the one or more linear PEI moieties comprises an average molecular weight of about 2500 Da.

[0102] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11-MUA-PEI polymer population comprises one or more 11- MUA moieties and one or more branched PEI moieties, each branched PEI moiety having an average molecular weight of about 600 Da, and a second 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and one or more linear PEI moieties, each linear moiety having an average molecular weight of about 2500 Da.

[0103] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, 14

[0104] ND 25-001

[0105] 501.112WO1 wherein the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11-MUA-PEI polymer population comprises one or more 11- MUA moieties and one or more branched PEI moieties, each branched PEI moiety having an average molecular weight of about 1800 Da, and a second 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and one or more linear PEI moieties, each linear PEI moiety having an average molecular weight of about 2500 Da.

[0106] In some embodiments, a medical implant coating disposed on a surface of the medical implant, the coating comprises a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i.a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core, forming a surface of the PhaNPs; and a plurality of 3-mercaptopropionic acid (3-MPA)-polyethylenimine (PEI) polymers conjugated to a surface of the PhaNPs, wherein the plurality of the 3-MPA-PEI polymers comprises one or more 3-MPA moieties and one or more branched and / or linear PEI moieties.

[0107] As noted, exemplary PhaNP comprise a silica inner core; one or more metal nanospheres disposed on surface of the silica core; and a plurality of CTF-PEI polymers conjugated to the one or more metal nanospheres.

[0108] In some embodiments, the silica core comprises silicon dioxide SiCE. The silica core may be synthesized, for example, the Stober process involving hydrolysis and condensation of silica precursor tetraethyl orthosilicate (Si(0Et)4, TEOS). The diameter of the resulting silica core 100 may be varied by varying the amount of ethanol, water, NH4OH solution, TEOS, and the material of the reaction vessel (see Stober et al., J. Colloid Interface Sci. 26, 62-69 (1968)).

[0109] In some embodiments, the metal nanosphere surrounding the silica core comprises one or more of potassium, sodium, barium, calcium, manganese, chromium, cadmium, iron, nickel, tin, lead, antimony, bismuth, arsenic, mercury, silver, gold, copper, zinc, platinum, palladium, magnesium, aluminum, and combinations thereof. In some embodiments, the surface layer metal comprises one or more of potassium, sodium, barium, calcium, manganese, chromium, cadmium, iron, nickel, tin, lead, antimony, bismuth, arsenic, mercury, silver, gold, copper, zinc, platinum, palladium, magnesium, aluminum, and combinations thereof.

[0110] In preferred embodiments, the PhaNPs comprise silica, silver, and gold nanoparticles may be fabricated as described in U.S. Patent Publication No. 2021 / 0252162 to Nallathamby et al.. In some embodiments, the disclosure provides for a nanoparticle comprising: a silica core; gold (Au) nanospheres disposed on a surface of the silica core; a silver (Ag) surface layer surrounding the gold nanosphere or silver alloyed into the gold nanosphere; and a plurality of CTF-PEO polymers (e.g., 11-MUA-PEI polymers) conjugated to the silver surface layer of the gold-silver nanospheres.

[0111] 15

[0112] ND 25-001

[0113] 501.112WO1 In some embodiments, the PhaNPs comprising: i. a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core, forming a surface of the PhaNPs; and a plurality of CTF-PEI polymers conjugated to a surface of the PhaNPs, wherein the plurality of the CTF-PEI polymers comprises one or more CTF moieties and one or more branched and / or linear PEI moieties.

[0114] In some embodiments, the PhaNPs comprising: i. a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core, forming a surface of the PhaNPs; and a plurality of 11-MUA-PEI polymers conjugated to a surface of the PhaNPs, wherein the plurality of the 11 -MUA-PEI polymers comprises one or more 11 -MUA moieties and one or more branched and / or linear PEI moieties.

[0115] In some embodiments, the PhaNPs comprise: i. a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core, forming a surface of the PhaNPs; and a plurality of 3-mercaptopropionic acid (3-MPA)-polyethylenimine (PEI) polymers conjugated to a surface of the PhaNPs, wherein the plurality of the 3-MPA-PEI polymers comprises one or more 3-MPA moieties and one or more branched and / or linear PEI moieties

[0116] Exemplary PhaNPs may be formed, for example, as described in U.S. Patent No. 12,161,725 to Nallathamby et al., U.S. Patent Publication No. 2010 / 0291537 to Souza et al., and PCT Patent Publication WO 2024 / 249419 to Nallathanby et al. In some embodiments, the PhaNPs may be formed, for example, by combining and stirring about 40 mL deionized water, about 400 pL NaOH (IM), about 3 mL sodium citrate dihydrate (68 nM), and about 1 mL THPC (85 mM) for approximately ten minutes at room temperature and then adding about 2 mL gold chloride (25 mM) and then covering and stirring for another approximately eight hours or more.

[0117] Silica cores may be prepared for adsorption by stirring for approximately eight hours or more with an additional about 15 mL ethanol, about 5 mL deionized water, and about 1 mL APTES and then centrifuging the SiCE-APTES cores for approximately thirty minutes at room temperature. The cores may then be separated from the supernatant, and the amino-functionalized nanoparticle pellets may be washed, sonicated (e.g., for 20 seconds at 40% amplitude), and centrifuged with about 10 mL ethanol twice at 9,000 rpm. The supernatant may then be discarded, and the residual pellets may be resuspended in 10 mL deionized water and sonicated (e.g., for 20 seconds at 40% amplitude).

[0118] In some embodiments, the gold nanospheres solution may be mixed into the SiCE-APTES cores solution, with a ratio of 1:2, for instance, and covered and stirred for approximately eight hours or more, resulting in gold nanospheres conjugated to (or immobilized on) the silica cores (referred to sometimes herein as SiO2@Au). The SiChOAu nanoparticle solution may then be 16

[0119] ND 25-001

[0120] 501.112WO1 centrifuged at 9,000 rpm for approximately fifteen minutes at room temperature, followed by one or more washing-sonication, centrifugation cycles in, for example, 10 mL deionized water. It should be appreciated that the foregoing is an exemplary formulation and protocol for synthesizing silica cores and gold nanospheres.

[0121] In some instances, the gold nanospheres are conjugated to the silica core so that the surface density of the gold nanospheres on the silica core is similar to the surface density of protein turrets on bacteriophages. As an illustrative example, a 45 nm silica core may include about a 0.0077 1 / nm2surface density of gold nanospheres, which is about 88% similar to the surface density of protein turrets of the bacteriophage SpV4 (0.0068 1 / nm2). In other instances, the distance between the gold nanospheres disposed on the silica core is such that the ratio of the silica core diameter to the distance between the gold nanospheres is within a range of 2 to 4. In other instances, the gold nanospheres are conjugated to the silica core such that the ratio of the diameter of the silica core to the distance between the gold nanospheres is greater than 4.

[0122] In some embodiments, the average number of silver coated gold nanospheres on the surface of the silica core is about 10 to about 80, about 20 to about 70, about 30 to about 60, or about 40 to about 50 silver coated gold nanospheres per 25 square nm of the silica core. In some embodiments, the average number of silver coated gold nanospheres on the surface of the silica core is about 35 to about 55 or about 40 to about 50 per 25 square nm of the silica core.

[0123] In some embodiments, the average number of CTF-PEI polymers (e.g., 11-MUA-PEI polymers) on the surface of the PhaNPs is about 100 polymers to about 900 polymers, about 150 to about 850, or about 200 to about 800. In some embodiments, the average number of CTF-PEI 600 polymers on the surface of the PhaNPs is about 650 to about 900, about 700 to about 900, or about 750 to about 850. In some embodiments, the average number of CTF-PEI 1800 polymers on the surface of the PhaNPs is about 100 to about 300, about 150 to about 250, or about 175 to about 225. In some embodiments, the average number of CTF-PEI 2500 polymers on the surface of the PhaNPs is about 50 to about 200, about 75 to about 175, or about 100 to about 150.

[0124] In some embodiments, the CTF moiety of the CTF-PEI polymers conjugated to the PhaNPs comprises one or more of In some embodiments, the CTF moiety comprises one or more of 11-mercaptoundecanoic acid, 3-Mercaptopropionic acid, mercaptoacetic acid, 2- mercaptobenzoic acid, 4-Mercaptobenzoic acid, 3 -Mercaptobenzoic acid, 4-Mercaptobutyric acid, 5-Mercaptopentanoic acid, Mercaptohexanoic acid, thiosalicylic acid, Thiophenecarboxylic acids (e.g., 2-mercapto-3-phenylpropionic acid), Cysteine, Homocysteine, and Penicillamine. The amines on the PEI can also be conjugated to thiol bearing organic molecules through reductive amination with an mercaptoaldehydes (e.g.,3-Mercapto-2-methylpentanal, 3-Mercaptohexanal, 17

[0125] ND 25-001

[0126] 501.112WO1 Mercaptoacetaldehyde) or mercaptoketone (e.g., 4-Mercapto-4-methyl-2-pentanone). Any NHS esters of CLICK reagents (e.g., DBCO-NHS, alkyne NHS ester, NHS Azide) can be utilized to introduce CLICKable functional groups on PEI by linking the CLICK reagent to the amine group of PEI through an NHS ester mediated amide bond formation. The complementary CLICK group on a thiol linker (e.g., Azide thiol to CLICK with DBCO, Azide thiol to CLICK alkyne through copper catalyzed reaction, DBCO-PEG-SH or DBCO-propanamide-thiol or DBCO- Thiol can be CLICKed to Azide) can be utilized to introduce a thiol group on the PEI as well. Isothiocyanates also react with amines. Thiol isothiocyanates such as EITC-PEG thiols (MW 200 or 400 or 600 or 800 or 1000 (www.biochempeg.com / product / EITC-PEG-SH.html) can also be utilized to introduce thiol functional group on PEI.

[0127] In some embodiments, the CTF moiety is 11-MUA. In some embodiments, the CTF moiety is 3-mercaptopropionic acid. In some embodiments, the average number of 11-MUA-PEI polymers on the surface of the PhaNPs is about 100 polymers to about 900 polymers, about 150 to about 850, or about 200 to about 800. In some embodiments, the average number of 11-MUA- PEI 600 polymers on the surface of the PhaNPs is about 650 to about 900, about 700 to about 900, or about 750 to about 850. In some embodiments, the average number of 11-MUA-PEI 1800 polymers on the surface of the PhaNPs is about 100 to about 300, about 150 to about 250, or about 175 to about 225. In some embodiments, the average number of 11-MUA-PEI 2500 polymers on the surface of the PhaNPs is about 50 to about 200, about 75 to about 175, or about 100 to about 150.

[0128] In some embodiments, a CTF-PEI polymer comprises about 1 to about 15 CTF molecules per PEI moiety, about 1 to about 12 CTF molecules per PEI moiety, about 1 to about 10 CTF molecules per PEI moiety, about 1 to about 8 CTF molecules per PEI moiety, or about 1 to about 5 CTF molecules per PEI moiety.

[0129] In some embodiments, a CTF-PEI polymer comprises about 1 to about 5 CTF molecules per linear PEI moiety, about 1 to about 3 CTF molecules per linear PEI moiety, or about 1 to about 15 CTF molecules per branched PEI moiety, about 2 to about 13 CTF molecules per branched PEI moiety, about 3 to about 12 CTF molecules per branched PEI moiety, or about 8 to about 12 CTF molecules per branched PEI moiety.

[0130] In some embodiments, an 11-MUA-PEI polymer comprises about 1 to about 10 11-MUA molecules per linear PEI moiety, or about 1 to about 3 11-MUA molecules per linear PEI moiety, or about 1 to about 15 11-MUA molecules per branched PEI moiety or about 2 11-MUA moieties to about 12 11-MUA molecules per branched PEI moiety. In some embodiments, an 11-MUA-

[0131] 18

[0132] ND 25-001

[0133] 501.112WO1 PEI polymer comprises about 8 to about 12 11-MUA molecules per branched PEI moiety or about 1 to about 5 11-MUA molecules per PEI moiety.

[0134] Some embodiments of PhaNPs have a silver coating alloyed to the surface of the gold nanospheres, which are disposed on the silica core. To accomplish this, in some instances, the SiO2@Au nanoparticle solution is mixed with 10 mM AgNOa and 10 mM freshly prepared hydroquinone solution. Different proportions of AgNOa and hydroquinone solution may be utilized for alloying different concentrations of silver on the gold nanospheres. For example, a low concentration of silver (referred to herein as LS) may utilize 3 mL AgNOa and 337.5 pL hydroquinone solution, a medium concentration of silver (referred to herein as MS) may utilize 3 mL AgNOa and 1.8 mL hydroquinone solution, and a high concentration of silver (referred to herein as HS) may utilize 7.2 mL AgNOa and 5.76 mL hydroquinone solution.

[0135] After mixing the SiO2@Au nanoparticle solution is mixed with a desired quantity of 10 mM AgNOa and 10 mM hydroquinone solution, in some instances, the mixed solution is covered completely with aluminum foil (e.g., to limit UV exposure) and stirred for approximately one hour. Then, the silver coating reaction may be stopped by centrifugation and discarding the supernatant. Various final concentrations of coated silver 104 are within the scope of this disclosure, depending on the size of the silica core, the gold nanospheres, and / or the intended concentration profile (e.g., LS, MS, HS). In some embodiments, the final silver coating concentration ranges from 5 pg / mL to 350.5 pg / mL. Furthermore, it will be appreciated that, in some embodiments, less than all of the gold nanospheres are coated with silver coating, whereas in other embodiments, all of the gold nanospheres are coated with silver coating. In some implementations, coating fewer than all of the gold nanospheres with silver coating provides PhaNPs that are more easily implemented into antibacterial applications for use within the human body (e.g., orthopedic implants). By way of illustrative example, the United States Environmental Protection Agency (EPA) has established a chronic oral Reference Dose (RfD) of 5 pg / kg / day for silver, which broadly translates to 250 pg to 750 pg per person per day (depending on the weight of the person). At least some of the presently disclosed PhaNPs include a maximum silver content of less than 200 pg (e.g., 189 pg / mL), with a predicted <1 pg / mL of silver ion leaching into solution, which is well below the EPA prescribed safety limit for silver exposure. Reducing the amount of silver coating on gold nanospheres of the ANPs may provide even lower levels of silver.

[0136] The resulting SiO2@Au@Ag PhaNPs may then be washed and centrifuged (e.g., two additional times in 10 mL deionized water) and stored in darkness until intended use. As discussed hereinbelow, the ANPs at least partially mimic the structure and / or dimensional characteristics of certain bacteriophages with icosahedral capsids (e.g., X174) and exhibit unexpected 19

[0137] ND 25-001

[0138] 501.112WO1 antibacterial properties. After all, adhesion of bacteriophages to a host bacterium is not usually antibacterial. Most phages require a metabolically active host to replicate their genomic nucleic acid and to form new virions. Thus, the infection following adhesion and the replication of phages within the bacterial host is typically the mode of bactericidal activity exhibited by phages.

[0139] In some embodiments, the PEI polymer moiety of the CTF-PEI polymers is prepared from low average molecular weight branched or linear moieties. Each moiety of the PEI branched or linear polymers comprise an average molecular weight (Da) of about 100 to about 5000, about 300 to about 3000, about 500 to about 2800, or about 600 to about 2500. In some embodiments, each moiety of the PEI branched or linear polymers comprise an average molecular weight (Da) of about 500 to about 3000, about 500 to about 2500, about 500 to about 2000, about 500 to about 1500, or about 500 to about 1500. In some embodiments, each moiety of the PEI branched or linear polymers comprise an average molecular weight (Da) of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, or about 3000.

[0140] In some embodiments, the PEI moiety of the CTF-PEI polymers is prepared from low average molecular weight branched moieties comprising individual moieties having an average molecular weight (Da) of about 500 to about 3000, or about 600 to about 2500. In some embodiments, the individual moieties of the PEI linear polymers comprise an average molecular weight (Da) of about 500 to about 3000, or about 1000 to about 2500. In some embodiments, the individual moieties of the PEI branched polymers comprise an average molecular weight (Da) of about 500 to about 700 or about 1700 to about 1900, or about 600 or about 1800. In some embodiments, the individual moieties of PEI linear polymers comprise an average molecular weight (Da) of about 1500 to about 3000, or about 2500. Preferably, the average molecular weight is determined by Light Scattering techniques, which are known in the art (for example, as described in U.S. Patent Publication No. 2020 / 0308022 to Bhat et al.) or via mass spectroscopy.

[0141] In other embodiments, the coating comprises polymers comprising a carboxy thiol functionalized (CTF) branched and / or linear PEI polymers. In some embodiments, the coating comprises CTF-PEI polymers having a molar ratio of about 5:1 to about 1:5 CTF:PEI. In some embodiments, the molar ratio of CTF:PEI is about 1: 1 to about 1:5, or about 1:1 to about 1:4, or about 1:1 to about 1:3, or about 1:1 to about 1:2, of about 1:1 to about 1:1.5, or about 1:1 to about 1:1.25. In other embodiments, the molar ratio of CTF:PEI is about 1:1.21. In other embodiments, the molar ratio of CTF:PEI is about 1:5, about 1:4.75, about 1:4.5, about 1:4.25, about 1:4, about 20

[0142] ND 25-001

[0143] 501.112WO1 1:3.75, about 1:3.5, about 1:3.25, about 1:3, about 1:2.75, about 1:2.5, about 1:2.25, about 1:2, about 1 : 1.75, about 1:1.5, about 1 : 1.25, or about 1 : 1. In some embodiments, the coating comprises polymers of CTF and branched and / or linear PEI polymers having a molar ratio of about 1:1.21 CTF:PEI.

[0144] In other embodiments, the coating comprises polymers comprising 11- Mercaptoundecanoic acid (11-MUA) and branched and / or linear PEI polymers. In some embodiments, the coating comprises 11-MUA-PEI polymers having a molar ratio of about 5: 1 to about 1:5 11-MUA:PEI. In some embodiments, the molar ratio of 11-MUA:PEI is about 1:1 to about 1:5, or about 1:1 to about 1:4, or about 1:1 to about 1:3, or about 1:1 to about 1:2, of about 1:1 to about 1:1.5, or about 1:1 to about 1:1.25. In other embodiments, the molar ratio of 11- MUA:PEI is about 1:1.21. In other embodiments, the molar ratio of 11-MUA:PEI is about 1:5, about 1:4.75, about 1:4.5, about 1:4.25, about 1:4, about 1:3.75, about 1:3.5, about 1:3.25, about 1:3, about 1:2.75, about 1:2.5, about 1:2.25, about 1:2, about 1:1.75, about 1:1.5, about 1:1.25, or about 1:1. In some embodiments, the coating comprises polymers of 11-MUA and branched and / or linear PEI polymers having a molar ratio of about 1 : 1.21 11-MUA:PEI.

[0145] In some embodiments, the coating comprises PhaNPs comprising CTF-PEI polymers, where the CTF-PEI polymers comprise polymers of two or more distinct polymer populations. For example, PhaNPs may comprise a first CTF-PEI polymer population comprising CTF-PEI polymers wherein the CTF-PEI polymers comprises CTF moieties and branched PEI polymer moieties, each branched PEI polymer moiety having an average molecular weight of about 600 Da, and a second CTF-PEI polymer population comprising CTF-PEI polymers wherein the CTF- PEI polymers comprise CTF moieties and linear PEI polymer moieties, each of the linear PEI polymer moiety having an average molecular weight of about 2500 Da. Other PhaNPs may comprise a first CTF-PEI polymer population comprising CTF-PEI polymers, wherein the CTF- PEI polymers comprise CTF moieties and branched PEI polymer moieties, each of the branched PEI polymer moieties having an average molecular weight of about 1800 Da, and a second CTF- PEI polymer population comprising CTF-PEI polymers, wherein the CTF-PEI polymers comprise CTF moieties and linear PEI polymer moieties, each of the linear PEI moiety having an average molecular weight of about 2500 Da.

[0146] In some embodiments, the coating comprises PhaNPs comprising 11-MUA-PEI polymers, where the 11-MUA-PEI polymers comprise polymers of two or more distinct polymer populations. For example, PhaNPs may comprise a first 11-MUA-PEI polymer population comprising 11-MUA-PEI polymers wherein the 11-MUA-PEI polymers comprises 11-MUA moieties and branched PEI polymer moieties, each branched PEI polymer moieties having an 21

[0147] ND 25-001

[0148] 501.112WO1 average molecular weight of about 600 Da, and a second 11-MUA-PEI polymer population comprising 11-MUA-PEI polymers wherein the 11-MUA-PEI polymers comprise 11-MUA moieties and linear PEI polymer moieties, each of the linear PEI polymer moieties having an average molecular weight of about 2500 Da. Other PhaNPs may comprise a first 11-MUA-PEI polymer population comprising 11-MUA-PEI polymers, wherein the 11-MUA-PEI polymers comprise 11-MUA moieties and branched PEI polymer moieties, each of the branched PEI polymer moieties having an average molecular weight of about 1800 Da, and a second 11-MUA- PEI polymer population comprising 11-MUA-PEI polymers, wherein the 11-MUA-PEI polymers comprise 11-MUA moieties and linear PEI polymer moieties, each of the linear PEI moieties having an average molecular weight of about 2500 Da.

[0149] In some embodiments, the average molecular weight of an individual CTF-PEI polymer is about 200 Da to about 5,000 Da, about 400 Da to about 4,000 Da, about 800 Da to about 3,300 Da, about 1000 Da to about 3,500 Da, about 1000 Da to about 3,100 Da, or about 1150 Da to about 3,100 Da. In some embodiments, the average molecular weight of an individual CTF-PEI polymer is about 1000 Da to about 4000 Da, about 1100 Da to about 3100 Da, about 1500 Da to about 2800 Da, or about 2800 Da, or about 1150 Da, or about 3100 Da. In some embodiments, the average molecular weight of an individual 11-MUA-PEI polymer is about 1000 Da to about 4000 Da, about 1100 Da to about 3100 Da, about 1500 Da to about 2800 Da, or about 2800 Da, about 1150 Da, or about 3100 Da. Methods for determining average molecular weight are known in the art, and include, but are not limited to static light scattering, size exclusion chromatography, and mass spectroscopy techniques.

[0150] As noted, the coating may be applied to the surface of a medical device, and in particular, a metal medical device having a metal surface. Non-limiting examples of medical devices are known in the art and also set forth herein. For example, in some embodiments, the medical device is one or more of a medical implant, a dental implant, and a surgical instrument. In some embodiments, the implants may be a prostheses, an implant as artificial substitutes for body parts, or materials inserted into tissue for functional, cosmetic, or therapeutic purposes. Prostheses can be functional, as in the case of artificial arms and legs; cosmetic, as in the case dermal filler; and therapeutic as in the case of implants surgically inserted or grafted into the body and intended to replace non-functioning organs. Exemplary medical implants further include vascular devices such as grafts (e.g., abdominal aortic aneurysm grafts, etc.), stents, catheters (including arterial, intravenous, blood pressure, stent graft, etc.), valves (e.g., polymeric or carbon mechanical valves,), embolic protection filters (including distal protection devices), vena cava filters, aneurysm exclusion devices, artificial hearts, cardiac jackets, and heart assist devices (including

[0151] 22

[0152] ND 25-001

[0153] 501.112WO1 left ventricle assist devices), implantable defibrillators, electro-stimulation devices and leads (including pacemakers, lead adapters and lead connectors), implanted medical device power supplies, peripheral cardiovascular devices, atrial septal defect closures, left atrial appendage filters, valve annuloplasty devices, mitral valve repair devices, vascular intervention devices, ventricular assist pumps, and vascular access devices (including parenteral feeding catheters, vascular access ports, central venous access catheters); surgical devices such as sutures of all types, anastomosis devices (including anastomotic closures), suture anchors, hemostatic barriers, screws, plates, clips, vascular implants, tissue scaffolds, cerebro-spinal fluid shunts, shunts for hydrocephalus, drainage tubes, catheters including thoracic cavity suction drainage catheters, abscess drainage catheters, biliary drainage products, and implantable pumps; orthopedic devices such as joint implants, acetabular cups, patellar buttons, bone repair / augmentation devices, spinal devices (e.g., vertebral disks and the like), bone pins, cartilage repair devices, and artificial tendons; dental devices such as dental implants and dental fracture repair devices; drug delivery devices such as drug delivery pumps, implanted drug infusion tubes, drug infusion catheters, and intravitreal drug delivery devices; ophthalmic devices such as scleral buckles and sponges, glaucoma drain shunts and intraocular lenses; urological devices such as penile devices (e.g., impotence implants), sphincter, urethral, prostate, and bladder devices (e.g., incontinence devices, benign prostate hyperplasia management devices, prostate cancer implants, etc.), urinary catheters including indwelling (“Foley”) and non-indwelling urinary catheters, and renal devices; synthetic prostheses such as breast prostheses and artificial organs (e.g., pancreas, liver, lungs, heart, etc.); respiratory devices including lung catheters; neurological devices such as neurostimulators, neurological catheters, neurovascular balloon catheters, neuro-aneurysm treatment coils, and neuropatches, splints, nasal tampons, ear wicks, ear drainage tubes, tympanostomy vent tubes, otological strips, laryngectomy tubes, esophageal tubes, esophageal stents, laryngeal stents, salivary bypass tubes, and tracheostomy tubes; oncological implants; and pain management implants.

[0154] Exemplary dental implants include root devices, such as those made from titanium, used to support restorations that resemble a tooth or group of teeth to replace missing teeth. Other examples of non-organic surfaces that may be coated or impregnated contemplated by the present invention include surfaces of medical supplies and medical equipment. Non-limiting examples of medical supplies and equipment include gloves (such as disposable gloves), gowns, pads, wheelchairs, stretchers, tables, swabs, sponges, sutures (such as silk sutures), bags, surgical supplies, and packaging materials for the packaging of sterile medical or hospital supplies. Further examples of non-organic surfaces include the surfaces of a sponge, wipe, pad, or mop.

[0155] 23

[0156] ND 25-001

[0157] 501.112WO1 Exemplary surgical instruments include Scalpels; Forceps: gullet forceps, bone clamping forceps, right angle forceps, abortion forceps, intestinal forceps, hemostatic forceps, artery forceps, towel forceps for scalp, ablation forceps, thread forceps, bulldog forceps for implantation, Fogarty blood vessel forceps, hemoclip forceps, gastric forceps, Eister forceps, caval forceps, duodenal forceps, bone forceps, stump appendix forceps, arthrosis forceps, tendon inducing forceps, thyroid forceps, lymphatic gland forceps, pleurolysis forceps, bone rongeur forceps, babcock forceps, multipurpose blood vessel forceps, curette forceps, connective tissue compressive forceps, mosquito forceps, Kiistner uterine safety forceps, blood vessel forceps, Pean forceps, Martin tenaculum forceps, drain forceps, Kocher's forceps, Muzeaux tenaculum forceps, Melabulldog forceps, Kelly's forceps, forceps with shank, thoraco- or laparo-scopic forceps, Allis forceps, Bulldog forceps, Mikulicz forceps, towel forceps, placenta forceps, etc.; Scalpel holders; Elevatoriums; Raspatriums; Hammers; Rongeurz; Spatulas: enteric spatula, cerebral spatula, nervous spatula, etc.; Luer; Tweezers; Retractors: Adson, Gelpi, Weitlaner, etc.; Mouth gags; Retractors: muscle retractor, Tensho type sharp retractor, nerve retractor, lung hemorrhage retractor, saddle retractor, extra-large width vastus muscle retractor, Diva retractor, Hohmann retractor, scapula retractor, ureteral retractor, tendon traction retractor, maxillary sinuses retractor, flat retractor, demar retractor, etc.; Kerrisons; Scissors: ophthalmic scissors, plastic surgery scissors, scanlan scissors, backward rib scissors, etc.; Drills; Needle holders: Mathieu, Seamed, Sarot, Rider, Webster, Sweden, etc.; Spreaders; Hooks: skin hook, French hook, etc.; Clips: Lenin Clip, etc.; Towels: cup, etc.; Retractors; Metal Petri dishes; Threads; Masks (for doctor, nurse, for ventilation of patients; Gloves; Surgical gowns; Gauzes; Needles: injection needle, aneurysm needle, cerebral ventricles tap needle, Deschamps aneurysm needle, etc.; Sondes; Beakers; Trays; Pus basins; Curettes: soft curette, dental bone curette; Rasoriums: dental rasorium, nerve rasorium, oral cleft rasorium, otolaryngological rasorium, etc.; Endoscopes: anal speculum, skull endoscope, nasal speculum, vaginal speculum, aural speculum, arthroscope, rectal speculum, capsule endoscope, etc.; Forceps: blood vessel forceps, Sweden forceps, Bergh forceps, microforceps, otolaryngological forceps, cushing forceps, dental forceps, hook forceps, bent nose DeBakey forceps long forceps, Adson forceps, Prince forceps, etc.; Tongue depressors; Suction tubes; Files; Mirrors; Stethoscopes; Scissors: Cooper, Mecchen, etc.; and Scissors (also see, for example, U.S. Patent No.8,877,222 to Aizawa et al.).

[0158] The disclosure also provides for methods of coating a surface of metal medical implant comprising the steps of polishing the surface of the metal medical implant; silanizing the surface of the metal medical implant; adhering a plurality of PhaNPs to the silanized surface; and

[0159] 24

[0160] ND 25-001

[0161] 501.112WO1 modifying the PhaNPs adhered to the surface of the medical implant with a plurality of 11-MUA- PEI polymers, wherein the PEI comprises a plurality of branched and / or linear PEI moieties.

[0162] Statements of certain embodiments of the invention.

[0163] 1. In a first embodiment, a coating is disposed on a surface of a medical implant, the coating comprising a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: a silica core; one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of carboxy thiol functionalized (CTF) -polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein each of the plurality of CTF-PEI polymers comprises one or more CTF moieties and one or more branched and / or linear PEI moieties.

[0164] 2. The coating of embodiment 1, wherein the carboxy thiol functionalized polyethyleneimines are represented by Formula I: each y is each independently 0-20.

[0165] 3. The coating of embodiment 1 or 2, wherein y is each independently 1-18.

[0166] 4. The coating of any one of embodiments 1-3, wherein y is each independently 5-15.

[0167] 5. The coating of any one of embodiments 1-4, wherein y is each independently 8-12.

[0168] 6. The coating of any one of embodiments 1-5, wherein the number average molecular weight of the carboxy thiol functionalized polyethylenimine of Formula I is less than 5000 Daltons.

[0169] 7. The coating of any one of embodiments 1-6, wherein the ethylenediamine moiety of each of R1, R2, and R3is recursive up to 100 times.

[0170] 25

[0171] ND 25-001

[0172] 501.112WO1 8. The coating of any one of embodiments 1-7, wherein the one or more CTF moieties comprises -CO(CI-C2O)SH or a -CO(thiophenyl).

[0173] 9. The coating of any one of embodiments 1-8, wherein the -CO(CI-C2O)SH, when present, is 11-mercaptoundecanoic acid, mercaptoacetic acid, 3-mercaptopropionic acid, 4- mercaptobenzoic acid, 3-mercaptobenzoic acid, or 6-mercaptohexanoic acid; and the - CO(thiophenyl), when present, is thiosalicylic acid.

[0174] 10. The coating of any one of embodiments 1-9, wherein the plurality of CTF moieties comprises 11-mercaptoundecanoic acid, and the plurality of CTF-PEI polymers comprise a plurality of 11-MUA-PEI polymers

[0175] 11. The coating of any one of embodiments 1-10, wherein the plurality of the CTF-PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:5 to about 5:1.

[0176] 12. The coating of any one of embodiments 1-11, wherein the plurality of the CTF-PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:1 to about 1:5.

[0177] 13. The coating of any one of embodiments 1-12, wherein the plurality of the CTF-PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:1 to about 1:3.

[0178] 14. The coating of any one of embodiments 1-13, wherein the plurality of the CTF-PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:1.21.

[0179] 15. The coating of any one of embodiments 1-14, wherein each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 250 Da to about 3500 Da.

[0180] 16. The coating of any one of embodiments 1-15, wherein each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 500 Da to about 3000 Da.

[0181] 17. The coating of any one of embodiments 1-16, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 500 Da to about 2000 Da.

[0182] 18. The coating of any one of embodiments 1-17, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da or about 1800 Da.

[0183] 19. The coating of any one of embodiments 1-18, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da.

[0184] 26

[0185] ND 25-001

[0186] 501.112WO1 20. The coating of any one of embodiments 1-19, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 1800 Da.

[0187] 21. The coating of any one of embodiments 1-20, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2000 to about 3000 Da.

[0188] 22. The coating of any one of embodiments 1-21, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2000 to about 2500 Da.

[0189] 23. The coating of any one of embodiments 1-22, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2500 to about 3000 Da.

[0190] 24. The coating of any one of embodiments 1-23, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2300 Da to about 2700 Da.

[0191] 25. The coating of any one of embodiments 1-24, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2500 Da.

[0192] 26. The coating of any one of embodiments 1-25, wherein the plurality of CTF-PEI polymers comprises at least two distinct CTF-PEI polymer populations, wherein a first CTF-PEI polymer population comprises one or more CTF moieties and a branched PEI moiety, the branched PEI moiety having an average molecular weight of about 500 Da to about 1800 Da, and a second CTF- PEI polymer population comprises one or more CTF moieties and a linear PEI moiety, the linear PEI moiety having an average molecular weight of about 2300 Da to about 2700 Da.

[0193] 27. The coating of any one of embodiments 1-26, wherein the plurality of CTF-PEI polymers comprises at least two distinct CTF-PEI polymer populations, wherein a first CTF-PEI polymer population comprises one or more CTF moieties and a branched PEI moiety, the branched PEI moiety having an average molecular weight of about 600 Da, and a second CTF-PEI polymer population comprises one or more CTF moieties and a linear PEI moiety, the linear PEI moiety having an average molecular weight of about 2500 Da.

[0194] 28. The coating of any one of embodiments 1-27, wherein the plurality of CTF-PEI polymers comprises at least two distinct CTF-PEI polymer populations, wherein a first CTF-PEI polymer population comprises branched PEI moieties having an average molecular weight of about 1800

[0195] 27

[0196] ND 25-001

[0197] 501.112WO1 Da, and a second CTF-PEI polymer population comprises linear PEI moieties having an average molecular weight of about 2500 Da.

[0198] 29. The coating of any one of embodiments 1-28, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 250 Da to about 5,000 Da.

[0199] 30. The coating of any one of embodiments 1-29, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 500 Da to about 4,000 Da.

[0200] 31. The coating of any one of embodiments 1-30, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 500 Da to about 3,750 Da.

[0201] 32. The coating of any one of embodiments 1-31, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 500 Da to about 3,500 Da.

[0202] 33. The coating of any one of embodiments 1-32, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 500 Da to about 3,250 Da.

[0203] 34. The coating of any one of embodiments 1-33, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 500 Da to about 3,000 Da.

[0204] 35. The coating of any one of embodiments 1-34, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 500 Da to about 2,500 Da.

[0205] 36. The coating of any one of embodiments 1-35, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 500 Da to about 2000 Da.

[0206] 37. The coating of any one of embodiments 1-36, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 150 Da to about 3,500 Da.

[0207] 38. The coating of any one of embodiments 1-37, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 600 Da to about 2,500 Da.

[0208] 39. The coating of any one of embodiments 1-38, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 400 Da to about 3,000 Da.

[0209] 40. The coating of any one of embodiments 1-39, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 600 Da to about 3,500 Da.

[0210] 41. The coating of any one of embodiments 1-40, wherein each of the plurality of CTF-PEI polymers comprises an average molecular weight of about 1000 Da to about 2,500 Da.

[0211] 42. The coating of any one of embodiments 1-41, wherein the coating has an average contact angle 9 of about 50° to about 65°.

[0212] 43. The coating of any one of embodiments 1-42, wherein the coating has an average contact angle 9 of about 55° to about 62°.

[0213] 44. The coating of any one of embodiments 1-43, wherein the carboxy thiol moiety of the carboxy thiol functionalized polyethylenimine polymers comprises mercaptoacetic acid, 3-

[0214] 28

[0215] ND 25-001

[0216] 501.112WO1 mercaptopropionic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, 6-mercaptohexanoic acid, 11-mercaptoundecanoic acid, or thiosalicylic acid.

[0217] 45. The coating of any one of embodiments 1-44, wherein the carboxy thiol moiety of the carboxy thiol functionalized polyethylenimine polymers comprises 11-mercaptoundecanoic acid or 3-mercaptopropionic acid.

[0218] 46. The coating of any one of embodiments 1-45, wherein the coating is disposed on a surface of a medical implant, the coating comprising: a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: i. a silica core; ii. one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of 11-mercaptoundecanoic acid (11-MUA)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein the plurality of 11-MUA-PEI polymers comprises one or more of 11-MUA moieties and one or more branched and / or linear PEI moieties.

[0219] 47. The coating of any one of embodiments 1-46, wherein the plurality of the 11-MUA-PEI polymers comprise a molar ratio of 11-MUA to PEI of about 1:5 to about 5:1.

[0220] 48. The coating of any one of embodiments 1-47, wherein the plurality of the 11-MUA-PEI polymers comprises a molar ratio of 11-MUA to PEI of about 1 : 1 to about 1:5.

[0221] 49. The coating of any one of embodiments 1-48, wherein the plurality of the 11-MUA-PEI polymers comprises a molar ratio of 11-MUA to PEI of about 1 : 1 to about 1:3.

[0222] 50. The coating of any one of embodiments 1-49, wherein the plurality of the 11-MUA-PEI polymers comprises a molar ratio of 11-MUA to PEI of about 1 : 1.21.

[0223] 51. The coating of any one of embodiments 1-50, wherein each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 250 Da to about 3500 Da.

[0224] 52. The coating of any one of embodiments 1-51, wherein each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 500 Da to about 3000 Da.

[0225] 53. The coating of any one of embodiments 1-52, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 500 Da to about 2000 Da.

[0226] 54. The coating of any one of embodiments 1-53, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da or about 1800 Da.

[0227] 29

[0228] ND 25-001

[0229] 501.112WO1 55. The coating of any one of embodiments 1-54, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da.

[0230] 56. The coating of any one of embodiments 1-55, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 1800 Da.

[0231] 57. The coating of any one of embodiments 1-56, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2000 to about 3000 Da.

[0232] 58. The coating of any one of embodiments 1-57, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2000 to about 2500 Da.

[0233] 59. The coating of any one of embodiments 1-58, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2500 to about 3000 Da.

[0234] 60. The coating of any one of embodiments 1-59, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2300 Da to about 2700 Da.

[0235] 61. The coating of any one of embodiments 1-60, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2500 Da.

[0236] 62. The coating of any one of embodiments 1-61, wherein the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11- MUA-PEI polymer population comprises one or more 11-MUA moieties and a branched PEI moiety, the branched PEI moiety having an average molecular weight of about 500 Da to about 1800 Da, and a second 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and a linear PEI moiety, the linear PEI moiety having an average molecular weight of about 2300 Da to about 2700 Da.

[0237] 63. The coating of any one of embodiments 1-62, wherein the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11- MUA-PEI polymer population comprises one or more 11-MUA moieties and a branched PEI moiety, the branched PEI moiety having an average molecular weight of about 600 Da, and a second 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and a linear PEI moiety, the linear PEI moiety having an average molecular weight of about 2500 Da.

[0238] 30

[0239] ND 25-001

[0240] 501.112WO1 64. The coating of any one of embodiments 1-63, wherein the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11- MUA-PEI polymer population comprises branched PEI moieties having an average molecular weight of about 1800 Da, and a second 11-MUA-PEI polymer population comprises linear PEI moieties having an average molecular weight of about 2500 Da.

[0241] 65. The coating of any one of embodiments 1-64, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 250 Da to about 4,000 Da.

[0242] 66. The coating of any one of embodiments 1-65, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 250 Da to about 3,750 Da.

[0243] 67. The coating of any one of embodiments 1-66, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 250 Da to about 3,000 Da.

[0244] 68. The coating of any one of embodiments 1-67, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 250 Da to about 2,800 Da.

[0245] 69. The coating of any one of embodiments 1-68, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 250 Da to about 2,500 Da.

[0246] 70. The coating of any one of embodiments 1-69, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 250 Da to about 2,000 Da.

[0247] 71. The coating of any one of embodiments 1-70, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 500 Da to about 3,500 Da.

[0248] 72. The coating of any one of embodiments 1-71, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 500 Da to about 3,300 Da.

[0249] 73. The coating of any one of embodiments 1-72, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 500 Da to about 2,800 Da.

[0250] 74. The coating of any one of embodiments 1-73, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 500 Da to about 2,500 Da.

[0251] 75. The coating of any one of embodiments 1-74, wherein each of the plurality of 11-MUA- PEI polymers comprises an average molecular weight of about 500 Da to about 2,000 Da.

[0252] 76. The coating of any one of embodiments 1-75, wherein the coating has an average contact angle 9 of about 50° to about 65°.

[0253] 77. The coating of any one of embodiments 1-76, wherein the coating has an average contact angle 9 of about 55° to about 62°.

[0254] 78. The coating of any one of embodiments, 1-77, wherein the one or more silver coated gold nanospheres on the surface of the silica core comprise an average of about 10 to about 80 silver coated gold nanospheres per 25 square nm of the silica core.

[0255] 31

[0256] ND 25-001

[0257] 501.112WO1 79. The coating of any one of embodiments, 1-78, wherein the one or more silver coated gold nanospheres on the surface of the silica core comprise an average of about 20 to about 70 silver coated gold nanospheres per 25 square nm of the silica core.

[0258] 80. The coating of any one of embodiments, 1-79, wherein the one or more silver coated gold nanospheres on the surface of the silica core comprise an average of about 30 to about 60 silver coated gold nanospheres per 25 square nm of the silica core.

[0259] 81. The coating of any one of embodiments, 1-80, wherein the one or more silver coated gold nanospheres on the surface of the silica core comprise an average of about 35 to about 55 silver coated gold nanospheres per 25 square nm of the silica core.

[0260] 82. The coating of any one of embodiments, 1-81, wherein the one or more silver coated gold nanospheres on the surface of the silica core comprise an average of about 40 to about 50 silver coated gold nanospheres per 25 square nm of the silica core.

[0261] 83. The coating of any one of embodiments 1-82, wherein the plurality of CTF-PEI polymers comprise about 1 to about 5 CTF moieties and a single branched PEI moiety.

[0262] 84. The coating of any one of embodiments 1-83, wherein the plurality of CTF-PEI polymers comprise about 6 to about 13 CTF moieties and a single branched PEI moiety.

[0263] 85. The coating of any one of embodiments 1-84, wherein the plurality of CTF-PEI polymers comprise about 1 to about 5 CTF moieties and a single linear PEI moiety.

[0264] 86. The coating of any one of embodiments 1-85, wherein the plurality of 11-MUA-PEI polymers comprise about 1 to about 5 11-MUA moieties and a single branched PEI moiety.

[0265] 87. The coating of any one of embodiments 1-86, wherein the plurality of 11-MUA-PEI polymers comprise about 6 to about 13 11-MUA moieties and a single branched PEI moiety.

[0266] 88. The coating of any one of embodiments 1-87, wherein the plurality of 11-MUA-PEI polymers comprise about 1 to about 5 11-MUA moieties and a single linear PEI moiety.

[0267] 89. The coating of any one of embodiments 1-88, wherein the plurality of CTF-PEI polymers comprises a ratio of CTF moieties to PEI moieties of about 1:1.21.

[0268] 90. The coating of any one of embodiments 1-89, wherein the plurality of 11-MUA-PEI polymers comprises a ratio of 11-MUA moieties to PEI moieties of about 1:1.21.

[0269] 91. A method for coating a surface of metal medical implant with the coating of any one embodiments 1-90 comprising the steps of: i) polishing the surface of the metal medical implant; ii) silanizing the surface of the metal medical implant; iii) adhering a plurality of PhaNPs to the silanized surface; iv) and modifying a surface of the PhaNPs adhered to the surface of the medical implant with a plurality of carboxy thiol functionalized (CTF)-PEI polymers, wherein the plurality of CTF-PEI polymers comprises a plurality of CTF moieties and a plurality of branched and / or

[0270] 32

[0271] ND 25-001

[0272] 501.112WO1 linear PEI moieties; and wherein the PhaNPs comprise: a) a silica core; and b) one or more silver coated gold nanospheres disposed on a surface of the silica core; and wherein the surface of the PhaNP comprises the one or more silver coated gold nanospheres.

[0273] 92. The method of embodiment 91, wherein the CTF is 11-mercaptoundecanoic acid.

[0274] 93. The method of embodiments 91 or 92, wherein the plurality of CT-PEI polymers comprise 11-MUA-PEI polymers comprising PEI moieties that are a) branched PEI moieties or b) linear PEI moieties, wherein the branched PEI moieties have an average molecular weight of about 500 Da to about 2000 Da and the linear PEI moieties have an average molecular weight of about 2000 Da to about 3000 Da.

[0275] 94. The method of any one of embodiments 91-93, wherein the CTF is 11- mercaptoundecanoic acid (11-MUA), and the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11-MUA-PEI polymer population comprises branched PEI moieties having an average molecular weight of about 600 or about 1800, and a second 11-MUA-PEI polymer population comprises linear PEI moieties having an average molecular weight of about 2500 Da.

[0276] 95. The coating of any of embodiments, 1-90, wherein the coating has a shared inflection of about 9 ~ 50° to about 62°, or about 62°.

[0277] 96. The coating of any of embodiments, 1-90, wherein the coating is dried on the surface of the medical device.

[0278] 97. A nanoparticle comprising: a silica core; one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of carboxy thiol functionalized (CTF)- polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein each of the plurality of CTF-PEI polymers comprises one or more CTF moieties and one or more branched and / or linear PEI moieties.

[0279] 98. The nanoparticle of embodiment 97, wherein the one or more CTF moieties comprises - CO(CI-C2O)SH or a -CO(thiophenyl).

[0280] 99. The nanoparticle of embodiment 97 or 98, wherein the -CO(CI-C2O)SH, when present, is 11-mercaptoundecanoic acid, mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, or 6-mercaptohexanoic acid; and the -CO(thiophenyl), when present, is thiosalicylic acid.

[0281] 100. The nanoparticle of any one of embodiments 97-99, wherein the plurality of CTF moieties comprises 11-mercaptoundecanoic acid, and the plurality of CTF-PEI polymers comprise a plurality of 11-MUA-PEI polymers

[0282] 33

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[0284] 501.112WO1 101. The nanoparticle of any one of embodiments 97-100, wherein the plurality of the CTF- PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:5 to about 5:1.

[0285] 102. The nanoparticle of any one of embodiments 97-101, wherein the plurality of the CTF- PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:1 to about 1:5.

[0286] 103. The nanoparticle of any one of embodiments 97-102, wherein the plurality of the CTF- PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:1 to about 1:3.

[0287] 104. The nanoparticle of any one of embodiments 97-103, wherein the plurality of the CTF- PEI polymers comprises a molar ratio of CTF moieties to PEI of about 1:1.21.

[0288] 105. The nanoparticle of any one of embodiments 97-104, wherein each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 250 Da to about 3500 Da.

[0289] 106. The nanoparticle of any one of embodiments 97-105, wherein each of the plurality of branched and / or linear PEI moieties comprise an average molecular weight of about 500 Da to about 3000 Da.

[0290] 107. The nanoparticle of any one of embodiments 97-106, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 500 Da to about 2000 Da.

[0291] 108. The nanoparticle of any one of embodiments 97-107, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da or about 1800 Da.

[0292] 109. The nanoparticle of any one of embodiments 97-108, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da.

[0293] 110. The nanoparticle of any one of embodiments 97-109, wherein each of the plurality of branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 1800 Da.

[0294] 111. The nanoparticle of any one of embodiments 97-110, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2000 to about 3000 Da.

[0295] 112. The nanoparticle of any one of embodiments 97-111, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2000 to about 2500 Da.

[0296] 34

[0297] ND 25-001

[0298] 501.112WO1 113. The nanoparticle of any one of embodiments 97-112, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2500 to about 3000 Da.

[0299] 114. The nanoparticle of any one of embodiments 97-113, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2300 Da to about 2700 Da.

[0300] 115. The nanoparticle of any one of embodiments 97-114, wherein each of the plurality of branched and / or linear PEI moieties is a linear polymer comprising an average molecular weight of about 2500 Da.

[0301] 116. The nanoparticle of any one of embodiments 97-115, wherein the plurality of CTF-PEI polymers comprises at least two distinct CTF-PEI polymer populations, wherein a first CTF-PEI polymer population comprises one or more CTF moieties and a branched PEI moiety, the branched PEI moiety having an average molecular weight of about 500 Da to about 1800 Da, and a second CTF-PEI polymer population comprises one or more CTF moieties and a linear PEI moiety, the linear PEI moiety having an average molecular weight of about 2300 Da to about 2700 Da.

[0302] 117. The nanoparticle of any one of embodiments 97-116, wherein the plurality of CTF-PEI polymers comprises at least two distinct CTF-PEI polymer populations, wherein a first CTF-PEI polymer population comprises one or more CTF moieties and a branched PEI moiety, the branched PEI moiety having an average molecular weight of about 600 Da, and a second CTF- PEI polymer population comprises one or more CTF moieties and a linear PEI moiety, the linear PEI moiety having an average molecular weight of about 2500 Da.

[0303] 118. The nanoparticle of any one of embodiments 97-117, wherein the plurality of CTF-PEI polymers comprises at least two distinct CTF-PEI polymer populations, wherein a first CTF-PEI polymer population comprises branched PEI moieties having an average molecular weight of about 1800 Da, and a second CTF-PEI polymer population comprises linear PEI moieties having an average molecular weight of about 2500 Da.

[0304] 119. The nanoparticle of any one of embodiments 97-118, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 250 Da to about 5,000 Da.

[0305] 120. The nanoparticle of any one of embodiments 97-119, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 500 Da to about 4,000 Da.

[0306] 121. The nanoparticle of any one of embodiments 97-120, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 500 Da to about 3,750 Da.

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[0308] ND 25-001

[0309] 501.112WO1 122. The nanoparticle of any one of embodiments 97-121, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 500 Da to about 3,500 Da.

[0310] 123. The nanoparticle of any one of embodiments 97-122, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 500 Da to about 3,250 Da.

[0311] 124. The nanoparticle of any one of embodiments 97-123, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 500 Da to about 3,000 Da.

[0312] 125. The nanoparticle of any one of embodiments 97-124, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 500 Da to about 2,500 Da.

[0313] 126. The nanoparticle of any one of embodiments 97-125, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 500 Da to about 2000 Da.

[0314] 127. The nanoparticle of any one of embodiments 97-126, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 150 Da to about 3,500 Da.

[0315] 128. The nanoparticle of any one of embodiments 97-127, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 600 Da to about 2,500 Da.

[0316] 129. The nanoparticle of any one of embodiments 97-128, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 400 Da to about 3,000 Da.

[0317] 130. The nanoparticle of any one of embodiments 97-129, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 600 Da to about 3,500 Da.

[0318] 131. The nanoparticle of any one of embodiments 97-130, wherein each of the plurality of CTF- PEI polymers comprises an average molecular weight of about 1000 Da to about 2,500 Da.

[0319] 132. The nanoparticle of any one of embodiments 97-131, wherein the one or more CTF moieties comprises -CO(CI-C2O)SH or a -CO(thiophenyl).

[0320] 133. The nanoparticle of any one of embodiments 97-132, wherein the -CO(CI-C2O)SH, when present, is 11-mercaptoundecanoic acid, mercaptoacetic acid, 3-mercaptopropionic acid, 4- mercaptobenzoic acid, 3-mercaptobenzoic acid, or 6-mercaptohexanoic acid; and the - CO(thiophenyl), when present, is thiosalicylic acid.

[0321] 134. The nanoparticles of any one of embodiments 97-133, wherein the CTF moiety is comprises one or more of 11-mercaptoundecanoic acid, mercaptoacetic acid, 3-mercaptopropionic acid, 2-mercaptobenzoic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, 4- mercaptobutyric acid, 5-mercaptopentanoic acid, mercaptohexanoic acid, thiosalicylic acid, thiophenecarboxylic acids, cysteine, homocysteine, penicillamine, mercaptoaldehydes or mercaptoketone, NHS esters of CLICK reagents and a complementary CLICK group on a thiol linker, isothiocyanates, and thiol isothiocyanates.

[0322] 36

[0323] ND 25-001

[0324] 501.112WO1 135. The nanoparticles of any one of embodiments 97-134, wherein the CTF moiety is 11- MUA.

[0325] 136. The nanoparticles of any one of embodiments 97-135, wherein the CTF moiety is 3- mercaptopropionic acid.

[0326] 137. A medical device comprising the coating of any of embodiments 1-90 and 96 disposed on the surface of the medical device.

[0327] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0328] EXAMPLES

[0329] Example 1. PhaNP and polyethylenimine polymer-based metal implant coating

[0330] Nanoparticle Characterization. This work utilizes phage-mimicking nanoparticles (PhaNPs) that structurally resemble bacteriophages up to 88%, taking advantage of the structure of the bacteriophage to exert an antibacterial effect towards antibiotic -resistant bacteria (Hopf et al., Nanoscale Advances, 2019, 1, 4812-4826). PhaNPs have proven to be effective against various strains of clinically relevant bacteria, exhibiting >99.9% inhibition in bacteria growth. TEM imaging revealed that the average diameter of the SiCE core was 22.91+2.74 nm (Figure 2A) while the average diameter of PhaNPs was 24.38+2.43 nm (Figure 2B). This confirmed that there was no significant change in the diameter of the SiCE core nanoparticle. The analysis of the TEM images also confirmed the presence of Au spheres on the SiCE core, showing that the synthesis process was successful. EDXS analysis showed distinct peaks for Si, Au and Ag, confirming their presence composition of the PhaNPs (Figure 2C). Zeta potential indicated the surface charge of the nanoparticles was 17.2 mV with a conductivity of 17 mS / cm.

[0331] Metal implant coupon characterization. SEM imaging was used to characterize the unmodified metal surface. Imaging showed that the surfaces of the metals were uniformly polished with no residual PhaNPs or cells prior to each surface modification (Figure 3A). EDXS analysis of the unmodified metals confirmed the implants as a stainless steel alloy of Fe, Cr and Ni with added Mo (Figure 3B). Stainless steel is one of the most widely used metals in implant materials, commonly used in orthopedic, dental and cardiovascular implants.

[0332] 37

[0333] ND 25-001

[0334] 501.112WO1 To confirm the success of implant modification, SEM imaging and FTIR analysis were carried out post-modification. The SEM imaging confirmed the presence of PhaNPs on the metal surface 3C), with the aggregated nanoparticles visible as black spots. FTIR analysis compared the modified and unmodified metal surfaces to further confirm the success of the surface modification by identifying the functional groups present on the modified surface. IR spectra for the unmodified metal surface showed no signal, while the modified surface had multiple distinct peaks 3D). The IR spectra of the PhANPs is consistent with other silica nanoparticle IR spectra with wide silica peaks centered around 1200 cm'1and a sharp peak at about 1550 cm'1PhANPs conjugated with polymers show a distinct peak at about 1650 cm'1which is likely a carbonyl peak resulting from the amide bond between the MUA and the polyethylenimine polymers. Contact angle measurements were analyzed for each modification done on the metal surface to determine the change in hydrophobicity (Table 1). The unmodified metal surface presented a slightly hydrophilic surface with contact angle of 80.49° ± 4.67°. Modification of the surface with PhaNPs showed a smaller contact angle indicating more hydrophilicity (67.43° ± 10.80°). Further modification of the PhaNP-modified metal surface with polymers led to greater hydrophilicity, with the smallest contact angle measurements exhibited on metals modified with MUA-EP (51.26° + 5.48°), and the mix of MUA-EP and MUA-PEI 1800 (55.71° + 11.48°).

[0335] Table 1. Contact Angle Measurements of Each Surface Modification.

[0336] Antibacterial activity against S. aureus USA300 and P. aeruginosa FRD1.

[0337] Cell viability of S. aureus USA300 and P. aeruginosa FRD1 was determined for the different surface modifications based off the Live / Dead assays. The results from the Live / Dead

[0338] 38

[0339] ND 25-001

[0340] 501.112WO1 assays were quantified using the ImageJ color segmentation plug-in, which allows for the segmentation of color in an image by pixels. Each individual pixel is matched to a color channel, and the number of pixels for each channel are then counted to achieve the area coverage of each channel the image. Red, green, yellow and background metal channels were chosen for each image. The red and yellow channels were summed together after the areas were determined. The Biotium Viability / Cytotoxicity Assay Kit for Bacteria uses the DNA dyes Ethidium Homodimer III which selectively permeate damaged cell membranes, and DMAO which permeable to intact membranes. Therefore, cells with damaged membranes will selectively fluoresce red, while cells with intact membranes will only fluoresce green. Cells may fluoresce yellow as a result of both dyes binding, indicating a damaged membrane. Membrane integrity has been established as an accepted criterion for cell viability. Green fluorescence can therefore be used as a measure of viable bacteria cells, while red or yellow fluorescence can indicate non- viable bacteria. The color segmentation tools divide the images by fluorescence to determine the presence of viable and non- viable.

[0341] Both 5. aureus USA 300 and P. aeruginosa FRD1 were shown to have viable cells on the unmodified metal implants as demonstrated by the Live / Dead assays (Figure 4A and C). SEM confirmed the presence and adhesion of each bacteria to the implant surface, indicating the fluorescence as coming from the bacteria membranes (Figure 4A and C). Therefore, the presence of both .S'. aureus USA 300 and P. aeruginosa FRD1 on the unmodified metals is confirmed. .S'. aureus USA300 exhibited relatively high cell viability on the unmodified metal and the PhANPs surfaces, with both having high area coverage of green fluorescence at 25.79% ± 18.90% and 66.20%° ± 13.42% respectively (Figure 4B). Compared to both the unmodified metal and the PhANPs surfaces, One-way ANOVA testing shows that the PhANPs + MUA-PEI 600, PhANPs + MUA-PEI 1800, PhANPs + MUA-PEI MUA-LP, PhANPs + MUA-PEI 600 + MUA-LP, and PhANPs + MUA-PEI 1800 + MUA-LP surface modifications have significantly less green fluorescence, with each of these modifications showing no green fluorescence (Figure 4B). This would indicate significantly less area where there are viable .S'. aureus cells, showing that these surface modifications reduce the ability for bacteria to grow. ANOVA testing does not show statistical differences in cell viability between the different polymers to indicate one surface modification as being the most successful at reducing cell viability (Figure 4B).

[0342] Analysis of red fluorescence indicates there is no statistical significance between any of the surface modifications in coverage of non-viable bacteria (Figure 4B). However, the PhANPs + MUA-PEI 600, PhANPs + MUA- PEI 1800, PhANPs + MUA-PEI LP, PhANPs + MUA-PEI

[0343] 39

[0344] ND 25-001

[0345] 501.112WO1 600 + MUA-LP, and PhANPs + MUA-PEI 1800 + MUA-LP surface modifications have significantly more area in which there was no bacteria, neither red or green fluorescence, on the metal compared to the unmodified and PhANPs surfaces (Figure 4B). This demonstrates that bacteria were significantly less successful at adhering to the surface of the metal implants in the surface modifications with the polymers. This is an important distinction for the mechanism of the polymers, as the polymer surface modifications do not indicate significant differences in red fluorescence which would indicate the ability for the modifications to kill the .S'. aureus USA300 (Figure 4B). Instead, the modification hydrophilicity could prove to be the mechanism of action that prevents the bacteria from adhering, with previous research demonstrating the hydrophilicity of positively charged polymer coatings as the principal mechanism against bacteria attachment and viability. The hydrophilic surface is likely to cause bacteria not to be able to attach to the surface causing them to slide off the surface. It has been previously confirmed that the biofilmforming bacteria attach better to the hydrophobic surface while hydrophilic surfaces repel bacterial adhesion. Contact angle measurements indicate that surfaces of the modifications with the polymers are more hydrophilic than the unmodified and PhANPs surfaces as indicated by the smaller angles (Table 1). This increase of surface hydrophilicity could therefore be the mechanism for lower cell viability.

[0346] The unmodified metal surface P. aeruginosa FRD1 showed relatively high area coverage of green fluorescence (25.51%° ± 5.88%), indicating cell viability on the unmodified surface. The PhANPs, PhANPs + MUA-PEI 600, PhANPs + MUA-PEI 1800, PhANPs + MUA-PEI LP, PhANPs + MUA-PEI 600 + LP, and PhANPs + MUA-PEI 1800 + LP surface modifications all had significantly less cell viability as (Figure 4D). This indicates that each surface modification is successful at reducing cell viability on the implants. Unlike with .S'. aureus USA300, the PhANPs surface modification is shown to be effective at reducing cell viability of P. aeruginosa FRD1. This is consistent with previous findings in which PhANPs were shown to slow the growth of P. aeruginosa FRD1 (Hopf et al., Nanoscale Advances, 2019, 1, 4812-4826), indicating the ability of the PhANPs surface modification to reduce cell viability. There is no statistical difference between any of the surface modifications in the red fluorescent area (Figure 4D). However, compared to the unmodified control, PhANPs, PhANPs + MUA-PEI 600, PhANPs + MUA-PEI 1800 PhANPs + MUA-PEI LP, PhANPs + MUA-PEI 600 + LP and PhANPs + MUA- PEI 1800 + LP all show significantly more area with no bacteria. Similarly to the .S'. aureus USA300, this indicates the lack of bacterial adhesion to the modified surfaces and could also be a result of the increased surface hydrophilicity.

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[0349] 501.112WO1 Biocompatibility of HaCaT and MG63. Cell viability was determined similarly to the bacteria by using the ImageJ color segmentation plug-in. The CytoCalccinTA Green in the Cell Imaging: Live or DcadT Cell Viability Assay Kit Green / Red Dual Fluorescence permeates intact cell membranes and will fluoresce green upon reaction with esterases. The propidium iodide in the assay can only permeate cell membranes that are no longer intact, making it selective to dead cells. Cells may fluoresce yellow as a result of both dyes binding, indicating a damaged membrane. Therefore, cells which only fluoresce green indicate viable cells while cells with any red fluorescence indicate non-viable cells. The color segmentation plug-in is used the same as in the bacteria to differentiate the area in which there are viable and non-viable cells.

[0350] SEM imaging confirms the presence of HaCaT cells on the metals with each surface modification (Figure 5A). The unmodified metal supported little HaCaT cell viability showing very little green fluorescence (0.05%° ± 0.03%), however, it did not kill the cells as there was no red fluorescence observed (Figure 5B). All the modifications were able to support viable cells as indicated by the (Figure 5B). The amount of green fluorescence is not statistically significant between any of the surface modifications, indicating that no surface modification is statistically significant at promoting HaCaT viability (Figure 5B). Red fluorescent area remains low on all surface modifications indicating that the modifications do not promote cell death (Figure 5B). The PhANPs surface modification has the highest red fluorescence area at 2.18%°+ 2.30%, although it is not statistically significant compared to any of the surface modifications. This indicates that none of the modifications contribute to cell death more than the others. Further, none of the surface modifications have significantly more area in which there is no cell adhesion (Figure 5B). However, although not statistically significant, the modified metal surfaces can be seen to have higher levels the HaCaT cell growth (Figure 5A). The Live / Dead images show more uniform coverage of the metal surface for each of the modifications, suggesting that there is the potential that these modifications promote HaCaT cell viability.

[0351] SEM imaging confirms the presence of MG63 cells on the surface of the metals with each modification (Figure 5C). The unmodified metal shows that MG63 cells are viable with 9.55%°+ 7.55% of the surface area being green fluorescence (Figure 5D). However, the PhANPs + MUA- PEI 600 + MUA-LP modification had lower cell viability (9.30%° ± 2.13%), with the MUA-LP modification showing significantly more green fluorescence and greater overall coverage as indicated by the less area of just metal (Figure 5D). This indicates that the MUA-LP modification promotes MG63 cell growth compared to the PhANPs + MUA-PEI 600 + MUA-LP modification and would be a preferred choice. However, there was no statistical significance that MG63 cells were more viable on MUA-LP compared to the control area, even though it was significant 41

[0352] ND 25-001

[0353] 501.112WO1 compared to PhANPs + MUA-PEI 600 + MUA-LP (Figure 5D). The other modifications were also not significant compared to control in the green fluorescence or no coverage channels (Figure 5D). Even though there was no statistical significance, just as in the HaCaT, it looks like the other modifications besides the PhANPs + MUA-PEI 600 + MUA-LP had better coverage (Figure 5C). On all the modifications there were few non-viable cells, with the PhANPs + MUA-PEI 600 modification having the most fluorescence at 1.05%° ± 1.48%, which indicates that all of the modifications do not cause cell death (Figure 5D). The belief that the polymers promoted HaCaT and MG63 cell viability even though there was no statistical significance is supported by previous research that showed metal implant modifications with polymers promoted cell growth. Also, the hydrophilic change in surface with the addition of the polymer conjugated nanoparticles could have affected mammalian cell growth. It has been previously proven that hydrophilic surfaces allow for better mammalian cell adhesion, promoting their growth. This explains the change in adherence for the bacteria, but continued adhesion from the mammalian cells when the surface became more hydrophilic. Overall, each surface modification was shown to be biocompatible with HaCaT and MG63 cells (Table 2 and Figure 8).

[0354] Table 2. Color segmentation data.

[0355] 42

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[0357] 501.112WO1

[0358] Green=alive; red=dead; metal=control, unmodified surface.

[0359] Example 2. Materials and Methods.

[0360] Materials. S. aureus USA300; P. aeruginosa FRD1; LB broth (Miller; Sigma- Aldrich); agar (Sigma- Aldrich); HACAT human skin keratinocytes; MG63 human osteoblasts; phosphate buffered saline (pH 7.4, IX, Cytiva); HyClone RPML1640 (Cytiva); HyClone Eagle’s Minimum Essential Medium (Cytiva); heat-inactivated fetal bovine serum (FBS; Peak Serum); trypsin- EDTA (Sigma- Aldrich); 200 proof ethanol (VWR); 20% w / v ammonium hydroxide (NH4OH; BDH); tetraethyl orthosilicate (TEOS; Sigma Aldrich, 99% GC grade); 3- aminopropyltriethoxy silane (APTES; Sigma Aldrich, 99%); sodium hydroxide (NaOH; Sigma Aldrich, >97%); sodium citrate dihydrate (C6H5Na3O7-2H2O; Sigma Aldrich, >99%); tetrakis(hydroxymethyl)phosphonium chloride (80% purity) (THPC; Sigma Aldrich, 80% in water); gold chloride (anhydrous; Sigma Aldrich); silver nitrate (AgNOa; Sigma Aldrich, 99% ACS grade); hydroquinone (Sigma Aldrich, >99% reagent grade); deionized water (DI water); 2- Propanol (Sigma Aldrich); 11 -Mercaptoundecyltrimethoxy silane (Gelest, Inc.); 4% buffered paraformaldehyde (VWR); Polyethylenimine branched 1800 (Thermo Scientific); Polyethylenimine branched 600 (Thermo Scientific), 11-Mercaptoundecanoic acid (Sigma Aldrich); Linear polyethylenimine 2500 (Polysciences); N,N’ -Diisopropylcarbodiimide (Sigma Aldrich); DMSO (Santa Cruz Biotechnology); Viability / Cytotoxicity Assay Kit for Bacteria Live and Dead Cells (Biotium); Cell Imaging: Live or Dead™ Cell Viability Assay Kit Green / Red Dual Fluorescence (AAT Bioquest).

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[0363] 501.112WO1 Silica core synthesis. Silica (SiC ) core nanoparticles were synthesized using the sol-gel (Stober) method of hydrolysis and condensation of TEOS, resulting in monodispersed silica nanoparticles with a diameter of 25 nm. 30 ml of ethanol, 6 ml of DI water and 1.8 ml of 20% NH4OH were added to a 100 ml glass beaker with a magnetic stir bar. The mixture was stirred at 250 rpm and to the solution, 1.125 ml of TEOS was added. The stir speed was increased to 550 rpm and the solution was stirred overnight (minimum of 12 h) at room temperature (22 °C). At room temperature, the SiO2 cores were centrifuged at 9000 rpm for 30 min to pellet the SiO2 nanoparticles. The pellet was washed with 10 ml ethanol, sonicated (pulse for 20 s at 40% amplitude) and rinsed again by centrifugation with 30 ml ethanol.

[0364] Silica core silanization. The silica cores nanoparticles were amine-functionalized by adding 25 ml ethanol, 5 ml DI water and 1 ml APTES with the nanoparticles in a 100 ml beaker. The solution was stirred overnight at 550 rpm at room temperature yielding SiCh-APTES cores. The solution was centrifuged at 9000 rpm for 30 min at room temperature to pellet the SiCh- APTES cores. The pellet was washed with 10 ml ethanol, sonicated (pulse for 20 s at 40% amplitude) and rinsed again by centrifugation with 30 ml ethanol. The supernatant was removed, and the pellet was resuspended in 10 ml of DI water, sonicated (pulse for 20 s at 40% amplitude) and moved to a clean 100 ml beaker with 20 ml DI water.

[0365] Gold nanosphere synthesis. Gold nanospheres with a diameter of 5 nm were synthesized using a modified procedure of alkaline reduction (Windels et al., The 1SME journal, 2019, 13, 1239-1251). Into a 250 ml beaker 43.16 ml DI water, 427 pl NaOH (1 M), 3.21 ml sodium citrate (68 mM) and 1.07 ml THPC (85 mM) was added. The solution was stirred at low speed (250 rpm) for at least 10 min at room temperature, followed by the addition of 2.14 ml of gold chloride (25 mM). The solution was stirred at 550 rpm at room temperature overnight in the dark.

[0366] Gold nanosphere deposition onto silica cores. The gold nanosphere solution was mixed with SiO2- APTES nanoparticle solution in a ratio of 1:2 and stirred overnight at 550 rpm in the dark, yielding SiO2@Au core-shell nanoparticles. The solution was centrifuged at 9000 rpm for 15 min at room temperature and the pellet was washed with 10 ml ethanol, sonicated (pulse for 20 s at 40% amplitude) and rinsed with 30 ml ethanol by centrifugation.

[0367] Silver coating gold nanospheres. Silver is alloyed onto gold nanospheres previously conjugated onto SiCE nanoparticles using the reduction reaction by hydroquinone (Gentry et al., Langmuir, 2009, 25, 2613-2621.). 30 ml of SiO2@Au nanoparticle solution was mixed with 7.2 ml 10 mM AgNOa and 5.76 ml 10 mM freshly prepared hydroquinone. The mixture was stirred at 500 rpm overnight in the dark, followed by halting the silver coating reaction by centrifugation and discarding the supernatant. SiO2@ Au@ Ag (PhANPs) pellet was washed with 30 ml DI water 44

[0368] ND 25-001

[0369] 501.112WO1 two times, sonicated (pulse for 20 s at 40% amplitude) and rinsed by centrifugation at 9000 rpm for 15 min at room temperature.

[0370] TEM imaging. SiCE core and PhANPs nanoparticle samples were prepared for TEM analysis by drop coating the dispersed sample onto a 300-mesh copper grid coated with amorphous carbon film. TEM images of the SiCh core and PhANPs nanoparticles were obtained on a JEOL 2011 microscope at an acceleration voltage of 200 kV. Further image analysis was carried out using ImageJ software. Diameters of 102 SiCh core nanoparticles and 102 PhANPs nanoparticles were measured from several TEM images selected and the average diameters of both samples were calculated.

[0371] Scanning Electron Microscopy (SEM) coupled with energy-dispersive X-ray spectrum (EDXS) analysis. To analyze the nanoparticle elemental content and confirm the presence of silica, gold and silver, SEM in conjugation with energy-dispersive X-ray spectrum (EDXS) analysis was carried out on Thermo Scientific Prisma E-SEM at an acceleration voltage of 15 kV, current of 70 pA and magnification of 100,000 x. A drop of resuspended PhaNP sample was placed onto an aluminum SEM stub and dried under vacuum. An EDXS map was created to scan over a site of PhANPs on the stub, with a count rate of 896 cps.

[0372] Unmodified metal implant surface characterization with SEM / EDXS. SEM imaging combined with EDXS analysis was carried out using the Thermo Scientific Prisma E-SEM to analyze the composition of the metals as well as image the metal surface after each step of modification. EDXS was performed on polished metals at an acceleration voltage of 15 kV, current of 0.14 nA and magnifications of 100,000 x. An EDXS map was created to scan over the metal surface, with a count rate of 1647 cps.

[0373] Metal implant surface preparation. The metal implant surfaces were polished with a Dremel tool using a Black Emery polishing compound until shiny. Polished metals were rinsed with 100% isopropanol and sonicated for 15 minutes.

[0374] Metal surface silane addition. The metals were cleaned using a UV-Ozone cleaner for 20 minutes at 25 °C. The ozone-treated metals were placed in a 100 ml beaker with a magnetic stir bar, 15 ml of 90% ethanol and 300 pl of 11 -mercaptoundecyltrimethoxy silane were added and the metals for 24 hours at 500 rpm. Following the silane addition process, the liquid was removed from the beaker, and the metals were sonicated for 15 minutes in 100% ethanol.

[0375] PhaNP modification of the metal surface. Silanized metals were placed in a 100 ml beaker with a magnetic stir bar. 1.5X PhaNP solution was prepared in 90% ethanol and 6 ml was added to the beaker with the metals. The solution was stirred for 24 hours at 500 rpm. The liquid was discarded and the metals were sonicated for 15 minutes in 100% ethanol .

[0376] 45

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[0378] 501.112WO1 Linking polymers onto PhaNP-modified metal implant surface. Polymer solutions were prepared by adding 11-mercaptoundecanoic acid, the PEI polymer, and N, N’ -di isopropylcarbodi imide in 0.909:1.1:50 molar ratio in 10 mL of DMSO. The PhaNP-modified metals were placed in a 50 ml beaker with a magnetic stir bar. The polymer solution (PEI6000.6 mL, PEI1800 0.8 mL and linear 0.6 mL) with 6 mL of 90% isopropanol was added to the beaker. If the modification was one with a mix a polymers, half of the specified volume was added for each polymer. The solution was stirred for 24 hours at 500 rpm, followed by discarding the liquid and washing the metals with 100% isopropanol twice. The washed metals were sonicated for 15 minutes.

[0379] SEM. SEM imaging was carried out at 10 kV and 0.24 nA. The polished metal surface was imaged at 170 x and 1500 x to confirm that the residual nanoparticles and cells had been removed during the metal polishing. Post-modification, the metal surface was also imaged at 170 x and 1500 x with the same settings to confirm that PhaNPs were present on the metal surface.

[0380] Fourier Transform Infrared (FTIR) spectroscopy FTIR spectroscopy was carried out to confirm the presence of functional groups on the metal surface, corresponding to the nanoparticles. A measurement of unmodified metal was taken prior to a measurement of the metal area containing PhANPs nanoparticles with polymer. All spectra were recorded in the 800-1800 cm-1 wavelength range.

[0381] Contact Angle measurements. Contact Angle determinations were made using the DMo- 701 KYOWA contact angle instrument. Measurements were made using the sessile drop technique and the tangent angle was measured at the three-phase contact point. The angle was calculated using the photography of the water and KYOWA interFAce Measurement and Analysis System FAMAS. One drop of water of volume of 2.0 pl was used for each trial. Each different surface modification was measured 4 times and the average of the measurements was calculated.

[0382] HaCaT human skin keratinocytes. Cells were cultured in Cytiva HyClone RPML1640 medium supplemented with 10% FBS at 37 °C in 5% CO2. Using a 12-well (3.8 cm2) cell-treated well plate, the metal coupons were added to an individual well before adding 100,000 HaCaT cells and 2 mL of full media. Metals were placed in ethanol for 30 minutes prior to being added to the well plate to sterilize them. The cells were incubated with the metals at 37 °C for 24 hours.

[0383] MG-63 Human Osteoblast-like Cells. Cells were cultured in Cytiva HyClone Eagle’s Minimum Essential Medium with 10% FBS at 37 °C in 5% CO2. Using a 12-well (3.8 cm2) cell- treated well plate, the metal coupons were added to an individual well before adding 100,000 MG63 cells and 2 mL of full media. Metals were placed in ethanol for 30 minutes prior to being

[0384] 46

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[0386] 501.112WO1 added to the well plate to sterilize them. The cells were incubated with the metals at 37 °C for 24 hours.

[0387] Mammalian cell viability assay. After incubation, the cells were imaged using the Cell Meter™ Cell Viability Assay Kit. Half of the media was replaced with assay buffer from a 10 mL stock containing buffer, 20 pl of each red-fluorescent propidium iodide and green-fluorescent CytoCalcein™ Green. The metals were incubated at 37°C in 5% CO2 for 30 minutes after the assay buffer was added. After incubation, the metals were washed in lx PBS solution and imaged using the Leica DM4000 B Fluorescence Motorized Microscope. Images were taken at lOx magnification using bright field, green fluorescence (FITC) and red fluorescence (RITC) channels.

[0388] Bacteria preparation. For antibacterial tests .S'. aureus USA300 and P. aeruginosa FRD1 were used. Bacteria stocks were stored at -80 °C before plating them on Luria Broth (LB) agar plate to isolate a single colony. The plates were incubated at 37 °C for 16-18 h, after which a single colony of each bacteria strain was picked and inoculated in liquid LB media at 37 °C for 12 h under continuous shaking (300 rpm). The 12 h inoculate was diluted to an OD600 of 0.05. 5 pl of the diluted bacteria inoculate was spread over a 60 mm LB agar plate. The metal coupons were sterilized in 100% ethanol for 30 minutes and placed surface side down onto the agar plates. The plates were incubated at 37 °C for 16 h.

[0389] Bacteria cell viability assay. After incubation, the bacteria were imaged using the Biotium Viability / Cytotoxicity Assay Kit for Bacteria. Metals were washed off in lx PBS solution before being added to 2.2 mL lx PBS, 2 pl red- fluorescent ethidium homodimer- 1 and 4 pl green- fluorescent calcein-AM. The metals were incubated in this solution for 15 minutes in the dark. After incubation, the metals were washed again in lx PBS and imaged using the Leica DM4000 B Fluorescence Motorized Microscope. Images were taken at lOx magnification using bright field, green fluorescence (FITC) and red fluorescence (RITC) channels.

[0390] SEM imaging fixed cells on metal implants. After cell viability assays, the metals with bacteria or mammalian cells were fixed in 4% paraformaldehyde buffered with IX PBS. The metals were coated with 7 nm carbon thread prior to imaging. Thermo Scientific Prisma E-SEM was used at 10-15 kV and 0.24-0.27 nA with magnifications of 100 x, 1000 x and 10,000 x.

[0391] Live / Dead Image Color Segmentation Analysis. Data analysis was completed using the Color Segmentation plug-in on ImageJ. The overlay images of the Live / Dead assays were used and 10 pixels of each color (green, red, yellow, and metal background) were selected for each channel. The Hidden Markov Model algorithm was used. 3 images from each surface modification were used, and the average percent area of each channel was determined for the given 47

[0392] ND 25-001

[0393] 501.112WO1 modification. The red and yellow channels area was summed together, and the standard deviations were combined to achieve the total red area. One-way ANOVA testing was completed comparing the differences in percent area coverage between the green, total red and background metal colors at a=0.05. / Polymer peak on IR is the carbonyl peak

[0394] Example 3. Antibacterial Activity Against Escherichia coli K-12.

[0395] To determine whether the anti-adhesive trends observed for S. aureus USA300 and P. aeruginosa FRD1 extend to additional Gram-negative species, E. coli K-12 were tested on identically prepared metal coupons (24 h, 37 °C). The Live / Dead staining and SEM workflow matched that described in Section 2.7.1-2.7.2, with the sole change that overnight LB cultures of E. coli were diluted to OD600 = 0.05 before plating. Quantitative color-segmentation analysis was performed as before.

[0396] Unmodified coupons again supported robust biofilm formation: discrete green (live) and red (dead) microcolonies were visible by fluorescence microscopy, and SEM revealed dense rod- shaped cells embedded in extracellular matrix .

[0397] PhaNP-only surfaces exhibited almost exclusively red fluorescence, indicating effective membrane disruption; however, SEM still showed a confluent layer of lysed bacterial debris adhering to the metal .

[0398] PhaNP@C3-PEI600 behaved similarly: Live / Dead assays showed near-complete killing, but cell corpses remained attached (Fig. 6C). In contrast, PhaNP@C3-PEI1800 reduced both colony viability and residual debris; only sparse cellular fragments were detected by SEM. Strikingly, the two most hydrophilic coatings — PhaNP@C3-LP and the mixed PhaNP@C3-LP + C3-PEI1800 formulation (9 ~ 51-56°) — eliminated detectable fluorescence signal and left the coupon surface essentially free of bacterial residue . Color-segmentation confirmed > 99.9 % empty area for both coatings, paralleling the performance previously seen against P. aeruginosa. Comparative Analysis Across Bacterial Species

[0399] Plotting % Empty area versus contact angle for the three test organisms (E. coli, P. aeruginosa, MRS A) revealed a shared inflection near 9 ~ 62°. Below this threshold, all coatings produced > 95 % empty area for every species; above it, residual attachment varied widely. This reinforces the concept of an optimal wettability window in which a stable hydration layer impedes initial adhesion of both Gram-positive and Gram-negative bacteria.

[0400] Mechanistic Implications

[0401] The persistence of cell debris on PhaNP and PhaNP@C3-PEI600 coupons — but not on the more hydrophilic LP-containing coatings — suggests that bactericidal activity alone is insufficient; 48

[0402] ND 25-001

[0403] 501.112WO1 effective anti-fouling requires simultaneous minimization of post-mortem adhesion. Linear PEI (LP) appears to confer a surface-energy landscape that discourages adsorption of both intact cells and lysed fragments, consistent with prior reports that low-density positive charge and high hydration favor fouling resistance.

[0404] Clinical Significance

[0405] Because E. coli is a common early colonizer in contaminated surgical sites, the ability of PhaNP@C3-LP and PhaNP@C3-LP + PEI1800 to both kill and repel E. coli widens the protective scope of these coatings beyond the opportunistic pathogens evaluated previously. Together with the cytocompatibility data, this supports advancing LP-containing formulations toward in vivo polymicrobial challenge models."

[0406] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0407] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

[0408] 49

[0409] ND 25-001

[0410] 501.112WO1

Claims

What is claimed is:

1. A coating disposed on a surface of a medical implant, the coating comprising a plurality of phage-mimicking nanoparticles (PhaNPs), the PhaNPs comprising: a silica core; one or more silver coated gold nanospheres disposed on a surface of the silica core; and a plurality of carboxy thiol functionalized (CTF)-polyethylenimine (PEI) polymers conjugated to the one or more silver coated gold nanospheres, wherein each of the plurality of CTF-PEI polymers comprises one or more CTF moieties and one or more branched and / or linear PEI moieties.

2. The coating of claim 1, wherein the one or more CTF moieties comprises -CO(Ci- C2O)SH or a -CO(thiophenyl).

3. The coating of claim 2, wherein the -CO(CI-C2O)SH, when present, is 11- mercaptoundecanoic acid, mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, or 6-mercaptohexanoic acid; and the -CO(thiophenyl), when present, is thiosalicylic acid.

4. The coating of claim 3, wherein the plurality of CTF moieties comprises 11- mercaptoundecanoic acid, and the plurality of CTF-PEI polymers comprise a plurality of 11- MUA-PEI polymers.

5. The coating of claim 4, wherein the plurality of 11-MUA-PEI polymers comprises a molar ratio of 11-MUA to PEI of about 1:5 to about 5:1.

6. The coating of claim 4, wherein the plurality of 11-MUA-PEI polymers comprise a molar ratio of 11-MUA to PEI of about 1:1 to about 1:5.

7. The coating of claim 1, wherein the one or more branched and / or linear PEI moieties comprise an average molecular weight of about 250 Da to about 3500 Da.

8. The coating of claim 1, wherein the one or more branched and / or linear PEI moieties comprise an average molecular weight of about 500 Da to about 3000 Da.50ND 25-001501.112WO19. The coating of claim 1, wherein the one more branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 500 Da to about 2000 Da.

10. The coating of claim 1, wherein the one or more branched and / or linear PEI moieties is a branched PEI moiety comprising an average molecular weight of about 600 Da or about 1800 Da.

11. The coating of claim 1, wherein the one or more branched and / or linear PEI moieties is a linear PEI polymer comprising an average molecular weight of about 2000 to about 3000 Da.

12. The coating of claim 1, wherein the one or more branched and / or linear PEI moieties is a linear PEI polymer comprising an average molecular weight of about 2500 Da.

13. The coating of claim 4, wherein the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and a branched PEI moiety having an average molecular weight of about 600 or about 1800, and a second 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and linear PEI moiety having an average molecular weight of about 2500 Da.

14. The coating of claim 3, wherein each of the plurality of 11-MUA-PEI polymers comprises and average molecular weight of about 250 Da to about 4,000 Da.

15. A method of coating a surface of a metal medical implant comprising the steps of: i) polishing the surface of the metal medical implant; ii) silanizing the surface of the metal medical implant; iii) adhering a plurality of phage mimicking nanoparticles (PhaNPs) to the silanized surface; iv) modifying a surface of the PhaNPs adhered to the surface of the medical implant with a plurality of carboxy thiol functionalized (CTF)-PEI polymers, wherein the plurality of CTF-PEI polymers comprises one or more CTF moieties and one or more branched and / or linear PEI moieties; and wherein the PhaNPs comprise:51ND 25-001501.112WO1a) a silica core; and b) one or more silver coated gold nanospheres disposed on a surface of the silica core; and wherein the surface of the PhaNP comprises the one or more silver coated gold nanospheres.

16. The method of claim 15, wherein the one or more CTF moieties of the CTF-PEI polymers comprises one or more of 11-mercaptoundecanoic acid, mercaptoacetic acid, 3- mercaptopropionic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, 6-mercaptohexanoic acid, and thiosalicylic acid.

17. The method of claim 16, wherein the one or more CTF moieties of the CTF-PEI polymers comprises 11-mercaptoundecanoic acid to form a plurality of 11-MUA-PEI polymers.

18. The method of claim 17, wherein each of the plurality of 11-MUA-PEI polymers comprise 11-MUA moieties and a PEI moiety that is a) a branched PEI moiety or b) a linear PEI moiety, wherein the branched PEI moiety has an average molecular weight of about 500 Da to about 2000 Da and the linear PEI moiety has an average molecular weight of about 2000 Da to about 3000 Da.

19. The method of claim 17, wherein each of the plurality of 11-MUA-PEI polymers comprises at least two distinct 11-MUA-PEI polymer populations, wherein a first 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and a branched PEI moiety having an average molecular weight of about 600 or about 1800, and a second 11-MUA-PEI polymer population comprises one or more 11-MUA moieties and linear PEI moiety having an average molecular weight of about 2500 Da.

20. A medical device comprising the coating of claim 1 disposed on the surface of the medical device.52ND 25-001501.112WO1

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